New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury.

Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.

Control of von Willebrand factor multimer size by thrombospondin-1.

Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury. Only the very large vWF multimers are effective in promoting platelet adhesion in flowing blood. A protein disulfide bond reductase in plasma reduces the average multimer size of vWF secreted by endothelial cells. This activity has been isolated from human endothelial cell conditioned medium and shown to be the trimeric glycoprotein, thrombospondin-1 (TSP-1). Incubation of purified TSP-1 with vWF resulted in formation of thiol-dependent complexes of TSP-1 and vWF, generation of new thiols in vWF, and reduction in the average multimer size of vWF. The ratio of the concentrations of TSP-1 and vWF in plasma reflected with average multimer size of vWF. The higher the plasma TSP-1/vWF molar ratio, the smaller the average vWF multimer size. In addition, administration of TSP-1 to mice resulted in reduction in the average multimer size of plasma vWF. Interaction of TSP-1 with vWF is mediated by TSP-1 type 1 properdin domains and the vWF A3 domain. These results indicate that TSP-1 regulates the multimeric size and therefore hemostatic activity of vWF.

Accumulation of PDGF B and cell-binding forms of PDGF A in the extracellular matrix.

PDGF is a powerful mitogen initially identified within platelets, but also shown to be produced by a wide variety of cell types. PDGF is encoded on two separate genes. These give rise to three polypeptides, PDGF B and two forms of PDGF A (SA and LA), resulting from alternative splicing of the PDGF A gene primary transcript. We report that in CHO cells transfected with PDGF gene constructs and producing moderate levels of PDGF homodimers, much of the PDGF LA and B produced, but little if any SA, is found in the matrix laid down beneath the cells. Immunoreactive PDGF in cells, and in matrix below expressing cells, was visualized by laser confocal microscopy. Western blotting of protein in matrix extracts, cell extracts, and secreted into the growth medium was used to demonstrate that the range of PDGF A polypeptides seen in the matrix was overlapping with those reported previously to be cell associated in cell types such as NIH3T3 and COS 7. This suggests that attachment to matrix or cell surface may be alternative fates for these polypeptides, with fate dependent on the characteristics of the producing cells. Immunoreactive PDGF A and B could be partially released by incubation of matrix material with heparin but not with other glycosaminoglycans. Digestion of matrix with chondroitin ABC lyase but not heparitinase or collagenase displaced some PDGF from its attachment sites. The results indicate attachment of PDGF to matrix proteoglycans, at least partly through the glycosaminoglycan moieties, and perhaps to additional components. The significance of matrix deposition for PDGF action is discussed.

Catalytic oligodeoxynucleotides define a key regulatory role for early growth response factor-1 in the porcine model of coronary in-stent restenosis.

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.

Immunology and clinical importance of antiphospholipid antibodies.

Having reviewed the literature on the association of aPL antibodies with clinical manifestations, it is clear that this group of autoantibodies are of considerable importance. The presence of aPL antibodies in some but not all individuals confers a risk of a clinical syndrome characterized by recurrent arterial or venous thrombosis, thrombocytopenia, hemolytic anemia, or positive Coombs' test, and in females, recurrent idiopathic fetal loss. In SLE, the risk is approximately 40%, compared with a risk of 15% in the absence of aPL antibodies. However, only one half of persons possessing these antibodies have SLE, and overall the risk is around 30%. In some circumstances, such as in chlorpromazine or infection-associated aPL antibodies, there appears to be no increased risk. At the other end of the spectrum are seen patients whose only clinical manifestations comprise features of this clinical syndrome, and this entity has been designated the primary antiphospholipid syndrome (PAPS). aPL antibodies are also important because they are not uncommon. They have been found frequently in women with idiopathic recurrent fetal loss (30%), in non-autoimmune patients with ischemic heart disease (20%), or venous thrombosis (up to 30%), or stroke (4-47%), and in chronic immune thrombocytopenia (30%). These autoantibodies can be detected using sensitive solid-phase immunoassays employing the CL antigen, or in appropriate coagulation tests to detect LA activity. These assays are simple to perform but require care in selection of the best test and in interpretation of results. Current tests do not distinguish between those persons at risk of the clinical events and those not at risk. Detection of specific isotypes (especially IgG) and antibody level may aid in such a designation. Treatment of aPL antibody-associated syndromes remains a controversial subject. Since thromboses are associated with significant morbidity and potential mortality, there is a good argument for long-term preventive antithrombotic therapy, at least for as long as the antibodies are detectable, in those patients in whom clinical complications have previously occurred. It is not generally recommended that this treatment be offered to individuals in whom aPL antibodies are detected but who have not suffered previous thromboses, since the risk of such events does not appear to be equal within a group of aPL antibody-positive persons. This particularly applies to pregnant women, since live births and uncomplicated pregnancies are observed regularly in the presence of aPL antibodies without specific treatment. A previous history of at least one unexplained, late fetal loss is considered a prerequisite before intervention in subsequent pregnancies.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of fish oil supplementation on glucose and lipid metabolism in NIDDM.

Fish oils, containing omega-3 fatty acids (omega 3FAs), favorably influence plasma lipoproteins in nondiabetic humans and prevent the development of insulin resistance induced by fat feeding in rats. We studied the effects of fish oils in 10 subjects (aged 42-65 yr) with mild non-insulin-dependent diabetes mellitus (NIDDM). Subjects were fed a standard diabetic diet plus 1) no supplementation (baseline), 2) 10 g fish oil concentrate (30% omega 3FAs) daily, and 3) 10 g safflower oil daily over separate 3-wk periods, the latter two supplements being given in radom order by use of a double-blind crossover design. At the end of each diet period, fasting blood glucose (FBG), insulin, and lipids were measured, and insulin sensitivity was assessed with a hyperinsulinemic-euglycemic clamp performed with [3-3H]glucose. FBG increased 14% during fish oil and 11% during safflower oil supplementation compared with baseline (P less than .05), whereas body weight, fasting serum insulin levels, and insulin sensitivity were unchanged. The absolute increase in FBG during each supplementation period correlated with the baseline FBG (fish oil, r = .83, P less than .005); safflower oil, r = .75, P = .012). Fasting plasma triglyceride levels decreased during fish oil supplementation in the 4 subjects with baseline hypertriglyceridemia (greater than 2 mM) but were not significantly reduced overall. There was no significant change in fasting plasma total, high-density lipoprotein, and low-density lipoprotein cholesterol levels. In summary, dietary fish oil supplementation adversely affected glycemic control in NIDDM subjects without producing significant beneficial effects on plasma lipids. The effect of safflower oil supplementation was not significantly different from fish oil, suggesting that the negative effects on glucose metabolism may be related to the extra energy or fat intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematologic correlates of left atrial spontaneous echo contrast and thromboembolism in nonvalvular atrial fibrillation.

OBJECTIVES: This study examined the relation between left atrial spontaneous echo contrast, hematologic variables and thrombo-embolism in patients with nonvalvular atrial fibrillation. BACKGROUND: Left atrial spontaneous echo contrast is associated with left atrial stasis and thromboembolism in patients with nonvalvular atrial fibrillation. However, its hematologic determinants in patients with nonvalvular atrial fibrillation are unknown. METHODS: Clinical, hematologic and echocardiographic variables were prospectively measured in 135 consecutive patients with nonvalvular atrial fibrillation undergoing transesophageal echocardiography. RESULTS: Patients with left atrial spontaneous echo contrast (n = 74, 55%) had an increased fibrinogen concentration (p = 0.029), platelet count (p = 0.045), hematocrit (p = NS) and left atrial dimension (p = 0.005). Multivariate analysis showed that left atrial spontaneous echo contrast was independently related to hematocrit (odds ratio = 2.24, p = 0.002), fibrinogen concentration (odds ratio = 2.08, p = 0.008) and left atrial dimension (odds ratio = 1.90, p = 0.004) but not platelet count. It was also associated with left atrial thrombus (n = 15, p = 0.001) and with recent embolism (n = 40, p < 0.001). In 40 clinically stable outpatients without previous embolism, left atrial spontaneous echo contrast was significantly related to hematocrit (p = 0.005), fibrinogen concentration (p = 0.035) and left atrial dimension (p = 0.029) but not to coagulation factor VII, D-dimer, erythrocyte sedimentation rate, platelet count, plasma beta-thromboglobulin, plasma glycocalicin or glycocalicin index. CONCLUSIONS: Left atrial spontaneous echo contrast in patients with nonvalvular atrial fibrillation is independently related to hematocrit, fibrinogen concentration and left atrial dimension, indicating a relatively hypercoagulable state in addition to stasis. These findings support the hypothesis that left atrial spontaneous echo contrast is due to erythrocyte aggregation. Hematologic factors may contribute to its association with thromboembolism.

Increased plasma beta-thromboglobulin in patients with coronary artery vein graft occlusion: response to low dose aspirin.

The therapeutic effect of aspirin on vein graft patency was studied in patients undergoing coronary artery bypass graft surgery. The study design enabled the prospective evaluation of the relation of platelet activation, as measured by plasma beta-thromboglobulin concentration, to subsequent coronary vein graft occlusion. Serial beta-thromboglobulin levels were measured in 105 patients randomized to receive aspirin (324 mg/day) or placebo beginning within 1 h after surgery. Graft patency was assessed angiographically at 1 week and 1 year after surgery. Of 49 patients receiving placebo, 17 (34.7%) had one or more graft occlusions, 6 early, 10 late and 1 with both early and late occlusion. Of 56 patients receiving aspirin, 7 (12.5%) had one or more occlusions, 3 early and 4 late (p less than 0.01). Preoperatively, the beta-thromboglobulin level in surgical patients (29 +/- 13.5 ng/ml) was significantly higher than that of 51 control subjects (22.6 +/- 11.1 ng/ml) (p less than 0.004). Plasma beta-thromboglobulin levels remained comparatively constant at 3 and 12 months after surgery in the 43 patients who had both samples available (p less than 0.001, r = 0.65). The reduction in beta-thromboglobulin concentration from the preoperative level to 12 months postoperatively was greater in the aspirin-treated group (p less than 0.001). Multivariate logistic regression analysis demonstrated a significant association between preoperative beta-thromboglobulin concentration and graft occlusion (p less than 0.02), and aspirin treatment was effective in preventing occlusion when adjusted for the preoperative beta-thromboglobulin level (p less than 0.005). Plasma beta-thromboglobulin concentrations are elevated in patients with coronary artery disease, suggesting ongoing platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular endothelium, haemostasis and thrombosis.

The vascular endothelium consists of a monolayer of cells. Integrity of the endothelium is essential for maintenance of blood fluidity as the subendothelium is composed of structures which rapidly activate platelets and coagulation. Recent research indicates that the endothelium is involved in other processes particularly vasoactivity, immune reactions and inflammatory processes. Much of the information has come from experiments using cells in culture and there has been considerable progress in defining synthesis and release of endothelial products involved. Of particular interest is the evidence for endothelial coagulant activity and exposure of binding sites for coagulation proteins, with the simultaneous suppression of antithrombotic activities, all of which can be brought about by inflammatory mediators. Despite the rapid progress in cell biology, clinical applications have tended to lag behind and there are still few aspects of endothelial cell metabolism and pathophysiology which can be precisely defined in patients with disorders in which the endothelium is without doubt actively involved.

Novel human PDGFA gene transcripts derived by alternative mRNA splicing.

The polymerase chain reaction (PCR) has been used to amplify sequences coding for the platelet-derived growth factor A chain (PDGFA) using mRNA populations derived from two transformed cell lines (a human osteosarcoma, U-2OS, and a human glioma, U-343) and from human umbilical vein cells. The primers used for PCR were designed to amplify both of the two transcripts previously reported for the PDGFA gene. These transcripts differ from each other by the presence or absence of sequences from a sixth exon located near the 3' end of the gene. The PCR procedure revealed not only these expected transcripts, but additional RNAs that were shown by cloning and sequencing to lack exon 2. These species were present at variable levels in the three cell types examined. We propose that this novel splicing pattern, generating mRNAs encoding truncated and non-functional polypeptides, signals an additional, post-transcriptional mechanism for modulation of PDGFA gene expression.

Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements.

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.

Phosphorylation sites for ribosomal S6 protein kinases in mouse 3T3 fibroblasts stimulated with platelet-derived growth factor.

Platelet release products and purified platelet-derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post-nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3-S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP-dependent and independent kinases.

Further observations on the effects of dietary fatty acid composition on platelet reactivity and blood coagulation in man and the influence of methodology on findings.

Eight human subjects were fed diets enriched in saturated fat (SF), or polyunsaturated fat (PUF) and after each dietary regimen the plasma heparinthrombin clotting time (HTCT) was determined. The HTCT of citrated plasma indicated reduced heparin-neutralizing activity (HNA) after PUF feeding compared with SF feeding. Platelet factor 4 (PF4) levels in the citrated plasma samples demonstrated an inverse correlation with the HTCT (r = 0.62). Experiments with purified PF4 indicated that the PF4 present in citrated plasma could only account for approximately 10% of the HNA. Plasma prepared in a manner which minimized in vitro release of platelet constituents contained significantly less PF4 after PUF feeding and indicated that most of the PF4 found in citrated plasma resulted from in vitro release. The factor Xa inhibitory activity of citrated plasma was not significantly altered by either of the dietary regimens.

Quantitation of platelet derived growth factor receptors on cells from human arterial segments.

The study of receptor expression in tissue containing multiple cell types presents a two-fold problem. Firstly, in situ studies are difficult to perform quantitatively and secondly, enzymatic disruption of the primary tissue results in the loss of cell surface receptors which may or may not be resynthesised. In vitro culture of cells following isolation may also alter receptor expression. To circumvent these problems, we have devised a reproducible non-enzymatic method for releasing endothelial and smooth muscle cells from human arterial segments and separating the mixture into constituent cell populations, in order to facilitate semi-quantitative receptor investigation by ELISA. The method involves the mechanical disruption of small artery segments and passage through progressively smaller sieves, resulting in a mixed population of single cells. Magnetic beads covalently attached to identifying lectins or antibodies are then used in a multistep procedure to give highly enriched cell populations. The dissociation/enrichment steps can be modified to select other cell types which may be present in the primary tissue, such as macrophages. We have used these preparations for locating and semi-quantitating PDGF and EGF receptors on the constituent cells of human umbilical artery preparations by ELISA using monoclonal receptor antibodies.

Quantitation of Fc gamma RII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization.

We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine.

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.

Reduction of von Willebrand factor by endothelial cells.

The haemostatic activity of plasma von Willebrand factor (vWF) is a function of multimer size. Only the large vWF multimers are effective in promoting platelet adhesion to a site of vascular injury. We observed that the conditioned medium of cultured human umbilical vein, human microvascular and bovine aortic endothelial cells contained an activity which reduced the average multimer size of plasma or purified vWF. The average multimer size of vWF produced endogenously by human umbilical vein endothelial cells was similarly reduced following secretion. The reducing activity was ablated by pre-treatment with heat or the thiol blocking agents. iodoacetamide, N-ethylmaleimide or E-64, but not by a range of specific serine-, cysteine-, aspartic-, or metalloproteinase inhibitors. Reduction in vWF multimer size was associated with formation of new thiols in vWF and there was no evidence for additional proteolytic processing of vWF. The reducing activity was associated with a protein with an anionic pi that binds heparin and contains reactive thiol(s). These results suggested that the interchain disulfide bonds that link the vWF homodimers near the N-termini were being reduced by a vWF reductase secreted by endothelial cells. In support of this hypothesis, incubation of vWF with the protein reductants, protein disulfide isomerase and thioredoxin, resulted in formation of new thiols in vWF and reduction in the average multimer size of vWF. These findings may have consequences for control of vWF haemostatic activity.

Effects of heparin and endothelial cell growth supplement on haemostatic functions of vascular endothelium.

Heparin in combination with endothelial cell growth factor (ECGF) affects physiological responses and growth of human umbilical vein endothelial cells (HUVEC). We have examined the effect of heparin, crude ECGF (endothelial cell growth supplement [ECGS]), or both on the basal and thrombin challenged output of metabolites by HUVEC. The supernatant and/or cell lysate was assayed for released prostacyclin, von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor and thrombospondin. Heparin modified release of all these metabolites when in combination with ECGS, and in general these responses were the opposite of those generated by inflammatory mediators such as interleukin-1. It has been postulated that heparin acts by potentiating the effect of ECGF, but heparin inhibited thrombospondin release and enhanced that of von Willebrand factor in the absence of ECGS, while ECGS alone inhibited release of plasminogen activator inhibitor. Thus, under our experimental conditions it would appear that heparin and crude ECGF can affect HUVEC independently of one another.

Anticardiolipin antibodies block the inhibition by beta 2-glycoprotein I of the factor Xa generating activity of platelets.

Antiphospholipid antibodies, defined either by lupus anticoagulant (LA) activity or positive anticardiolipin immunoabsorbent assay (ACA) are associated with a predisposition to thromboses, recurrent fetal loss or thrombocytopenia. The mechanisms for these predispositions remain undefined. We have enriched immunoglobulin fractions from two patient plasmas to obtain antibodies with LA activity but no ACA, or conversely, with ACA positivity but no LA, in order to investigate in vitro characteristics which might explain a thrombotic propensity. beta 2-glycoprotein I (beta 2-GPI), the plasma cofactor required for ACA binding to negatively charged phospholipid, has previously been shown to inhibit prothrombinase generation in the presence of activated platelets (8). We now report that beta 2-GPI, at physiological concentrations, inhibits the generation of factor Xa in the presence of activated gel-filtered platelets. Further, ACA interferes with this inhibition, resulting in protracted, unopposed factor Xa generation. This interference with beta 2-GPI, a natural anticoagulant component of plasma, is potentially prothrombotic. LA immunoglobulins behave differently and inhibit factor Xa generation in a manner similar to beta 2-GPI. These findings provide the basis for a previously unsuspected mechanism for thrombosis in patients with aPL.

Complete covalent structure of human platelet factor 4.

The amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with beta-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.