| 1. |
- Lanke, E., et al.
(författare)
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Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1
- 2004
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Ingår i: Journal of Thrombosis and Haemostasis. - : Wiley-Blackwell. - 1538-7933 .- 1538-7836. ; 2:11, s. 1918-1923
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Tidskriftsartikel (refereegranskat)abstract
- Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.
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| 2. |
- Malm, Karl, et al.
(författare)
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Antithrombotic and anticoagulant effects of wild type and Gla-domain mutated human activated protein C in rats.
- 2007
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Ingår i: Thrombosis Research. - : Elsevier BV. - 1879-2472 .- 0049-3848. ; 120:4, s. 531-539
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Tidskriftsartikel (refereegranskat)abstract
- The antithrombotic and anticoagulant effects of recombinant wild type (WT) and mutated human activated protein C (hAPC) were investigated using a rat model of arterial thrombosis. Recent in vitro studies using human plasma have shown enhanced anticoagulant effects of hAPC by mutagenesis of either loop 148 in the serine protease domain or of the Gla domain. The Gla-domain mutant QGNSEDY-hAPC (=H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y) was found to be particularly active as an anticoagulant. We now combined the two mutations to create the variant QGNSEDY-hAPC:B148 and investigated the in vivo effects of this variant as well as of QGNSEDY-hAPC and WT hAPC using a rat model of arterial thrombosis. In vitro clotting experiments using rat plasma demonstrated WT hAPC to be inefficient, whereas both mutant hAPC variants yielded distinct dose dependent anticoagulant effects. In the arterial injury model, a segment of the left common carotid artery was opened longitudinally. An endarterectomy was performed and the arteriotomy was closed, whereafter the vessel was reperfused and the patency rate determined after 31 min. Three treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of 0.8 mg/kg WTor mutant hAPC or vehicle only. The ex vivo clotting times of plasma drawn 3 min after the injections, as compared to baseline clotting times, were approximately doubled by QGNSEDY-hAPC and tripled by QGNSEDY-hAPC:B148 infusions, while WT APC had little effect. Compared to the control group, none of the hAPC preparations had significant antithrombotic effect or increased arteriotomy bleeding.
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| 3. |
- Malm, Karl, et al.
(författare)
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Human activated protein C variants in a rat model of arterial thrombosis
- 2008
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Ingår i: Thrombosis Journal. - : Springer Science and Business Media LLC. - 1477-9560. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Activated protein C (APC) inhibits coagulation by degrading activated factor V (FVa) and factor VIII (FVIIIa), protein S (PS) functioning as a cofactor to APC.METHODS: By mutagenesis of the vitamin K-dependent Gla domain of APC, we have recently created an APC variant having enhanced anticoagulant activity due to increased affinity for negatively charged phospholipid membranes. In the present study, the potential antithrombotic effects of this APC variant, and of a variant APC that is additionally mutated in the serine protease domain, have been evaluated in a blind randomized study in a rat model of arterial thrombosis. In this model, we have previously found the combination of bovine APC and PS to be highly antithrombotic. Four treatment groups each containing 10 rats were, in a blind random fashion, given intravenous bolus injections of wild-type or mutant variants of APC (0.8 mg/kg) together with human PS (0.6 mg/kg) or human PS (0.6 mg/kg) alone. A control group with 20 animals where given vehicle only.RESULTS: A trend to increased patency rates was noted in a group receiving one of the APC variants, but it did not reach statistical significance.CONCLUSION: In conclusion, administration of human APC variants having enhanced anticoagulant efficacy together with human PS in a rat model of arterial thrombosis did not give an efficient antithrombotic effect. The lack of effect may be due to species-specific differences between the human protein C system and the rat hemostatic system.
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| 4. |
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| 5. |
- Malm, Karl, et al.
(författare)
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Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S.
- 2003
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Ingår i: Thrombosis and Haemostasis. - 0340-6245. ; 90:2, s. 227-234
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Tidskriftsartikel (refereegranskat)abstract
- The antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, where-as bAPC combined with bPS prolonged the clotting time con-siderably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent antithrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.
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| 6. |
- Ahmad, Abrar, et al.
(författare)
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Evaluation of Expression Level of Apolipoprotein M as a Diagnostic Marker for Primary Venous Thromboembolism
- 2018
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Ingår i: Clinical and Applied Thrombosis/Hemostasis. - : SAGE Publications. - 1938-2723 .- 1076-0296. ; 24:3, s. 416-422
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Tidskriftsartikel (refereegranskat)abstract
- Recently, decreased levels of apolipoprotein M (ApoM) were shown to be associated with higher risk of recurrent venous thromboembolism (VTE) in male patients. However, the role of ApoM in primary VTE is unknown. We aimed in our study to analyze the plasma levels of ApoM in patients with VTE in order to evaluate the diagnostic importance of ApoM in primary VTE. A total of 357 patients with suspected first episode of VTE were recruited prospectively in the SCORE study. Plasma samples from 307 patients were available for quantifying the plasma levels of ApoM in patients with VTE using sandwich enzyme-linked immunosorbent assay method. Among the whole population, plasma levels (mean [standard deviation]) of ApoM were not significantly different between patients with VTE (0.72 [0.20]) and non-VTE patients (0.72 [0.16]), P = .99. Similarly, in regression analyses, no significant association of ApoM plasma levels with the risk of VTE was found on univariate (odds ratio [OR] =1.0, 95% confidence interval [CI] 0.21-4.84, P = .99) and multivariate analysis (OR = 1.25, 95% CI = 0.19-8.34, P = .819) after adjusting for age, body mass index, and smoking. Moreover, results did not differ significantly after stratification of data according to sex ( P > .05). In this study, our results do not suggest a diagnostic role for ApoM plasma levels in patients with primary VTE. Moreover, the current study suggests that role of ApoM as a risk factor may differ for primary VTE and recurrent VTE in male patients.
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| 7. |
- Ahmad, Abrar, et al.
(författare)
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Identification of polymorphisms in Apolipoprotein M gene and their relationship with risk of recurrent venous thromboembolism
- 2016
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Ingår i: Thrombosis and Haemostasis. - 0340-6245. ; 116:3, s. 41-432
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (ApoM) plasma levels have been reported to be associated with risk of venous thromboembolism (VTE) recurrence. However, the role of genetic alterations in the ApoM gene in VTE recurrence remains unknown. The aim of this study was to identify genetic aberrations in ApoM gene in VTE recurrence and their role in prediction of VTE recurrence in a prospective follow-up study of 1465 VTE patients. During follow-up, 156 (10.6 %) patients had VTE recurrence. First screening of whole ApoM gene was performed by Sanger's sequencing in selected age and sex matched non-recurrent and recurrent patients (n=95). In total six polymorphisms were identified and two polymorphisms (rs805297 and rs9404941) with minor allele frequency (MAF) ≥5 % were further genotyped in the whole cohort by Taqman PCR. ApoM rs805297 polymorphism was significantly associated with higher risk of VTE recurrence in males but not in females on both univariate (p= 0.038, hazard ratio = 1.72, confidence interval = 1.03-2.88) and on multivariate analysis adjusted with mild and severe thrombophilia, family history, location and acquired risk factors for VTE. However, ApoM rs9404941 polymorphism showed no significant association with risk of VTE recurrence in all patients as well as in different gender groups. Moreover, ApoM rs805297 and rs9404941 polymorphisms were not associated with the ApoM plasma levels. In conclusion, for the first time we have sequenced whole ApoM gene in VTE and identified six polymorphisms. ApoM rs805297 was significantly associated with higher risk of VTE recurrence in male but not in female patients.
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| 8. |
- Ahnström, Josefin, et al.
(författare)
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Activated protein C cofactor function of protein S: a novel role for a gamma-carboxyglutamic acid residue
- 2011
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 117:24, s. 6685-6693
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Tidskriftsartikel (refereegranskat)abstract
- Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the omega-loop that mediates Ca2+-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is gamma-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca2+ coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function. (Blood. 2011;117(24):6685-6693)
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| 9. |
- Ahnström, Josefin, et al.
(författare)
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HDL Stimulates apoM Secretion.
- 2010
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Ingår i: Protein & Peptide Letters. - 0929-8665. ; Jul 1, s. 1285-1289
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (apoM) in human plasma is mainly associated with HDL. A retained signal peptide anchors apoM to the lipoproteins. To investigate the role of the signal peptide in the transfer of apoM from the synthesizing cell to the lipoproteins, wildtype apoM cDNA and the Q(22)A mutant, introducing a signal peptidase cleavage site, were used to stably transfect HEK293 cells, which intrinsically do not express apolipoproteins. When cultured under serum-free conditions, wildtype apoM was, in contrast to Q(22)A, poorly secreted. Addition of serum or purified HDL stimulated secretion of wildtype apoM, which was recovered in the medium incorporated in HDL. The liver cell line HepG2, which synthesizes HDL, was cultured under serum-free conditions and found to secrete apoM as part of an HDL-like particle. In conclusion, due to its retained signal peptide, apoM is poorly secreted unless HDL is either coexpressed or added to the culture medium.
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| 10. |
- Ahnström, Josefin, et al.
(författare)
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Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M
- 2007
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 48:8, s. 1754-1762
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of pre beta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound ( dissociation constant 5 2-3 mu M), whereas other tested substances ( e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.
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