A peptide from human semenogelin I self-assembles into a pH-responsive hydrogel.

The peptide GSFSIQYTYHV derived from human semenogelin I forms a transparent hydrogel through spontaneous self-assembly in water at neutral pH. Linear rheology measurements demonstrate that the gel shows a dominating elastic response over a large frequency interval. CD, fluorescence and FTIR spectroscopy and cryo-TEM studies imply long fibrillar aggregates of extended ß-sheet. Dynamic light scattering data indicate that the fibril lengths are of the order of micrometers. Time-dependent thioflavin T fluorescence shows that fibril formation by GSFSIQYTYHV is a nucleated reaction. The peptide may serve as basis for development of smart biomaterials of low immunogenicity suitable for biomedical applications, including drug delivery and wound healing.

The influence of a new low molecular weight sulphated polysaccharide from mussel broth (Org 30016) on haemostasis.

The effect on haemostasis of a new low molecular weight sulphated polysaccharide (4300 dalton) from mussel broth (Org 30016) was investigated. In an APT time test system it had an anticoagulant effect corresponding to 30-40% of that of heparin. It had no antithrombin effect. It exerted its effect only in test systems containing phospholipids. It did not prolong haemostatic plug formation time in the rabbit mesenteric microcirculation as did heparin. Formation of ex vivo Chandler thrombi was significantly inhibited. The practical interest of the substance comes from the combination of an antithrombotic effect and little influence on primary haemostasis.

Properties and catabolism of heat treated antithrombin III concentrate.

Heat treatment is employed to diminish the transmission of hepatitis when blood products are administered. It is possible that such a procedure could reduce the biological activity of the proteins and induce changes in structure and aggregation state. We have therefore made in vitro and pharmacokinetic studies of heat treated antithrombin III (AT III) concentrate using both radiolabelled and non-labelled preparations. The purification, heat treatment and the radiolabelling procedures did not induce any changes in the AT III molecules with exception of a decrease in heparin affinity in about 10% of the molecules. The in vivo studies using 125I AT III showed that the fractional catabolic rate was increased and the half-life was shortened by about 20-25% compared to our previous studies on non-heat treated AT III concentrate. Our present findings indicating a mean half-life of 3.0 days are quite comparable to studies by others on non-heat treated AT III, however.

High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction.

The anticoagulant vitamin K-dependent protein S interacts with the complement regulatory protein C4b-binding protein (C4BP), both in purified systems and in plasma. The concentrations of these proteins in plasma are approximately equimolar (0.3 microM) and 30-40% of protein S in plasma is found in the noncomplexed state. Only the uncomplexed form of protein S displays anticoagulant activity and studies have shown that patients with a selective deficiency of free protein S have a high incidence of thrombosis. In this study, we report that the protein S-C4BP interaction is at least 100-fold tighter in the presence of Ca2+ than in EDTA. The KD in the presence of Ca2+ was estimated with a gel filtration technique to be less than 5 x 10(-10) M, whereas in the presence of EDTA, it was approximately 100-fold higher. Ca2+ titration experiments suggested that the Ca2+ sites which function in the protein S-C4BP interaction are of high affinity which, in turn, suggests that they may be independent of the gamma-carboxyglutamic acid region and may be present in the epidermal growth factor-like domains of protein S. The high affinity of the protein S-C4BP interaction in the presence of Ca2+ suggested that virtually all of the protein S in whole blood should be complexed with C4BP. However, even though the protein S-C4BP interaction in Ca2(+)-containing serum was shown to have the same high affinity as in purified systems, approximately 30-40% of the protein S in serum was free. These results appear best explained by the presence of a third component in whole blood which regulates the protein S-C4BP interaction, keeping approximately 30-40% of circulating protein S in its free, functionally anticoagulant form. It is speculated that persons with little free protein S may be deficient in this hypothetical third component.

Platelet-associated IgG in childhood idiopathic thrombocytopenic purpura: measurements on intact and solubilized platelets and after gammaglobulin treatment.

An immunoradiometric assay was developed for determining platelet-associated IgG (PAIgG) both on intact and solubilized platelets. In 20 healthy subjects PAIgG was 0.28 +/- 0.20 ng/10(6) platelets and 2.2 +/- 1.1 ng/10(6) platelets on intact and on solubilized platelets, respectively. 13 children with acute ITP all had increased concentrations of PAIgG, but no correlation was found between the severity of thrombocytopenia and PAIgG concentrations, either on intact or on solubilized platelets. Neither was there any correlation of the increase in PAIgG between intact and solubilized preparations. 3 out of 4 children who received high-dose i.v. gammaglobulin showed a concomitant normalization of the platelet count and of the PAIgG concentration both on intact and lyzed platelets. The remaining child did not respond to gammaglobulin and continued to have abnormal PAIgG.

Inhibition of platelet prothrombinase activity by a lupus anticoagulant.

Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.

A Swedish family with abnormal antithrombin III.

An abnormal variant of antithrombin III is reported in a young male with deep vein thrombosis. The heparin cofactor, progressive thrombin inhibition, and factor Xa inactivation are decreased. The abnormality seems to be a mutation which is transmitted in an autosomaldominant way. The half-life and fractional catabolic rate of 125I antithrombin III concentrate is the same in this patient as in patients with the classic type of antithrombin III deficiency and in a control.

Antithrombin III concentrate: its catabolism in health and in antithrombin III deficiency.

The catabolism of purified radiolabelled antithrombin III (AT III) concentrate was studied in two normals and two patients with congenital AT III deficiency both alone and combined with warfarin. The radiolabelling with iodine monochloride did not change the quality of the concentrate. The half-life varied between 3.4 and 4.8 days. No difference between normals and patients with congenital deficiency in non-acute stage could be observed in the catabolic parameters; nor was there any influence with warfarin.

A novel semi-synthetic sulphated polysaccharide with potent antithrombin activity.

A mucopolysaccharide was isolated from mussel broth. After sulphation, an electrophoretically homogenous product was obtained with a molecular weight of approximately 40 000 daltons and a sulphur content of about 12%. The sulphated polysaccharide (S-Lim) displayed an anticoagulant activity in a thrombin test system with human plasma. Unlike heparin, the anticoagulant effect of S-Lim was observed also in a thrombin-fibrinogen clotting system in the absence of AT III. Complete inhibition of the effect of 1.0 mg S-Lim was achieved with 1.5 mg protamine. In an activated partial thromboplastin time test system the anticoagulant activity of 1.0 mg S-Lim corresponded to about 40 iu of heparin. S-Lim was also found to inhibit thrombin-induced platelet aggregation. Furthermore, S-Lim inhibited the thrombin-dependent activation of factor XIII in human plasma. S-Lim did not affect various tests systems measuring factor Xa activity. It is concluded that this new sulphated mucopolysaccharide acts as a pure antithrombin with a potency corresponding to 40--50 iu heparin/mg S-Lim. In contrast to heparin and other heparinoids it does not require the presence of AT III.

Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes.

BACKGROUND: Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. METHODS AND RESULTS: In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. CONCLUSIONS: These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.

The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA).

BACKGROUND: The sperm chromatin structure assay (SCSA) provides an objective assessment of sperm chromatin integrity, which is essential for normal sperm function. SCSA is valuable as a fertility marker in epidemiological studies and in the clinical situation. Little is known about the impact of testicular and post-testicular function on SCSA parameters. METHODS: Ejaculates from 278 military conscripts of median age 18.1 (range 18-21) years were included. Levels of reproductive hormones, the length of the CAG repeat of the androgen receptor gene, sperm concentration, abstinence period and biochemical parameters of epididymal and accessory sex gland secretions were correlated to the SCSA parameters, DNA fragmentation index (DFI) and highly DNA stainable (HDS) cells. RESULTS: Negative correlations were found between sperm concentration and DFI (r = -0.119, P = 0.049) and HDS (r = -0.513, P < 0.0001). DFI was negatively correlated with levels of estradiol (r = -0.19, P = 0.002) and free testosterone (r = -0.13, P = 0.03). DFI also correlated positively with abstinence time (r = 0.17, P = 0.005), and with seminal concentrations of fructose (r = 0.18, P = 0.004) and zinc (r = 0.12, P = 0.04). CONCLUSIONS: Sex steroid production, spermatogenic function, abstinence time and seminal vesicle function appear to impact on sperm chromatin integrity and thereby on sperm fertilizing capacity. These findings may improve present understanding of the pathophysiology of male infertility.