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Introduction: Venous thrombosis is the leading cause of pregnancy-related maternal morbidity and mortality. The thrombosis risk is increased by caesarean section and blood loss, though underlying mechanisms of these prothrombotic changes remain unknown., Materials and Methods: This prospective study recruited 50 pregnant women at term undergoing elective caesarean section at University Hospital Magdeburg, Germany. Blood loss during surgery was correlated with the changes in total protein S, full length TFPI (TFPI fl ), prothrombin, the endogenous thrombin potential (ETP) and resistance to activated protein C (APCsr) determined via calibrated automated thrombography., Results: Mean blood loss was 506 ml (95%CI: 456 to 557 ml). Total protein S was 0.63 (95%CI: 0.60 to 0.67) U/ml preoperatively, decreased by 14.8% after caesarean section and almost normalised five days later. TFPI fl was 0.47 (95%CI: 0.41 to 0.53) U/ml before, remained unchanged immediately after and increased by 11.5% five days after surgery. Prothormbin was 1.10 (95%CI: 1.03 to 1.16) U/ml preoperatively, reduced by 10.4% immediately after and increased again five days after caesarean section, exceeding the preoperative values by 4.4% (-0.7 to 9.6). The ETP decreased by 3.9%, whereas the APCsr increased by 37.0% immediately after caesarean section. The changes in total protein S, prothrombin, thrombin generation and APC resistance showed a trend to be more pronounced in the subgroups with higher blood loss., Discussion: Moderate blood loss during caesarean section hardly reduces thrombin generation but aggravates pregnancy-induced APC resistance and combined deficiency of TFPI and protein S, which can account for the increased thrombosis risk in early puerperium., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Decreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification., Methods: A dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems., Results: Monovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma., Conclusion: In this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation., Summary: Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation., (© 2017 International Society on Thrombosis and Haemostasis.)

Essentials The C-terminus of tissue factor pathway inhibitor (TFPIα) binds to the B-domain of factor V (FV). The functional consequences of this interaction were investigated in plasma and model systems. The TFPIα C-terminus inhibited thrombin generation in plasma, but not in the presence of FVa. The TFPIα C-terminus inhibited FV activation by preventing cleavage at Arg 1545 ., Summary: Background Factor V (FV) is a carrier and a cofactor of the anticoagulant protein tissue factor pathway inhibitor-α (TFPIα), whose basic C-terminus binds to an acidic region in the B-domain of FV. Proteolysis of FV at Arg 709 , Arg 1018 and Arg 1545 by activated FX (FXa) or thrombin removes the B-domain, and converts FV into a procoagulant cofactor (activated FV [FVa]) of FXa in the prothrombinase complex. However, retention of the acidic region in partially activated FV makes prothrombinase activity susceptible to inhibition by TFPIα. Objective/Methods To investigate the effect of the TFPIα C-terminal peptide (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. Results TFPIα C-term inhibited tissue factor-initiated and FXa-initiated thrombin generation in a dose-dependent manner. Failure to inhibit thrombin generation in FV-depleted plasma reconstituted with FVa indicated that the peptide effect was mediated by the acidic region of FV, and was localized at the level of FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited both FV activation and prothrombinase activity. Western blot analysis showed that the peptide impaired cleavage at Arg 1545 by both thrombin and FXa. The inhibition was stronger for FV-short, which binds TFPIα with higher affinity. Similar results were obtained with full-length TFPIα. Conclusions Cleavage of FV at Arg 1545 , which abolishes the anticoagulant properties of FV and commits FV to the procoagulant pathway, is inhibited by binding of the TFPIα C-terminus to the FV acidic region. Possible targets of this new anticoagulant function of TFPIα are low-abundance FV(a) species retaining the acidic region., (© 2016 International Society on Thrombosis and Haemostasis.)

BACKGROUND AND PURPOSE Endothelin-1 (ET-1) causes long-lasting vasoconstrictions. These can be prevented by ET(A) receptor antagonists but are only poorly reversed by these drugs. We tested the hypothesis that endothelin ET(A) receptors are susceptible to allosteric modulation by endogenous agonists and exogenous ligands. EXPERIMENTAL APPROACH Rat isolated mesenteric resistance arteries were pretreated with capsaicin and studied in wire myographs, in the presence of L-NAME and indomethacin to concentrate on arterial smooth muscle responses. KEY RESULTS Endothelins caused contractions with equal maximum but differing potency (ET-1 = ET-2 > ET-3). ET-1(1-15) neither mimicked nor antagonized these effects in the absence and presence of ET(16-21). 4(Ala) ET-1 (ET(B) agonist) and BQ788 (ET(B) antagonist) were without effects. BQ123 (peptide ET(A) antagonist) reduced the sensitivity and relaxed the contractile responses to endothelins. Both effects depended on the agonist (pK(B): ET-3 = ET-1 > ET-2; % relaxation: ET-3 = ET-2 > ET-1). Also, with PD156707 (non-peptide ET(A) antagonist) agonist-dependence and a discrepancy between preventive and inhibitory effects were observed. The latter was even more marked with bulky analogues of BQ123 and PD156707. CONCLUSIONS AND IMPLICATIONS These findings indicate allosteric modulation of arterial smooth muscle ET(A) receptor function by endogenous agonists and by exogenous endothelin receptor antagonists. This may have consequences for the diagnosis and pharmacotherapy of diseases involving endothelins., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI)., Objective: The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation., Results: At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n)., Conclusions: Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.

A mutation (W501S) in the vitamin K-dependent gamma-glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC-W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495-513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d-OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high-affinity site for d-OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the KM for vitamin K hydroquinone in wild-type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.