Background
Coagulation factor V (FV), present in plasma and platelets, has both pro‐ and anticoagulant functions.
Objective
We investigated an FV‐deficient patient (FV:C 3%, FV:Ag 4%) paradoxically ...presenting with recurrent venous thrombosis (11 events) instead of bleeding.
Methods/Results
Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet‐poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet‐rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC‐cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon) that favors a “closed conformation” of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5‐fold) unfavorable kinetic parameters (Km, Vmax) of prothrombin activation, but also a lower rate of APC‐catalyzed inactivation in the presence of protein S.
Conclusions
FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid‐binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.
Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. During thrombotic events, TFPI is proteolytically inactivated by ...neutrophil elastase while bound to neutrophil extracellular traps (NETs). Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine to citrulline and is crucial for NET formation.
Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines.
Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied using western blotting and mass spectrometry. Binding of TFPIα to PAD4 was investigated using a solid-phase assay. Functional consequences were investigated by factor Xa inhibition and thrombin generation assays.
Nanomolar PAD4 amounts eliminated factor Xa inhibition by TFPIα. A citrullinated mutant Kunitz 2 domain did not inhibit factor Xa. Citrullination of TFPIα was found to be time- and concentration-dependent. Immunoprecipitation of citrullinated proteins from whole blood after neutrophil activation suggested the presence of TFPIα. Negatively charged phospholipids inhibited citrullination and truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were less efficiently citrullinated by PAD4. TFPIα bound to PAD4 with nanomolar affinity and involved the basic C-terminus. Thrombin generation in TFPI-deficient plasma demonstrated reduced anticoagulant activity of citrullinated TFPI. Mass spectrometry demonstrated citrullination of surface-exposed arginine residues in TFPIα after incubation with PAD4.
Full-length TFPIα is sensitive to citrullination by PAD4, which causes loss of factor Xa inhibition. This process may play a role in the increased thrombosis risk associated with inflammation.
•Protein arginine deiminase 4 (PAD4) is involved in the formation of neutrophil extracellular traps (NETs); tissue factor pathway inhibitor (TFPI) can bind to NETs.•Coincubation of TFPIα with PAD4 led to loss of TFPI activity and an increase of citrulline residues.•PAD4 can interact with TFPIα. Modification of arginine 107 led to loss of antifactor Xa activity.•Citrullination of TFPIα could contribute to the thrombotic risk associated with inflammatory disease.
OBJECTIVE—Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced ...thrombus formation in vitro and in vivo.
METHODS AND RESULTS—High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIIbβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3.
CONCLUSIONS—These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.
Summary
In a study population consisting of healthy men (n=8), women not using oral contraceptives (OC) (n=28) and women using different kinds of OC (n=187) we used calibrated automated thrombography ...(CAT) in the absence and presence of added activated protein C (APC) to compare parameters that can be obtained from thrombin generation curves, i.e. lag time, time to peak, peak height and endogenous thrombin potential (ETP). Both with and without APC, plasmas of OC users exhibited the shortest lag time and time to peak, and the highest peak height and ETP. In the absence of APC none of these parameters differed between users of OC containing different progestogens. In contrast, in the presence of APC shorter lag times and time to peak, and higher peak height and ETP were observed in plasma of users of gestodene-,desogestrel-,drospirenone- and cyproterone acetate-containing OC than in plasma of users of levonorgestrel-containing OC. The ETP determined in the absence of APC (ETP-APC) had no predictive value for the APCsr (r=0.11; slope 0.9×10
–3
; 95%CI: –0.1×10
–3
to 2.0×10
–3
) whereas the ETP measured in the presence of APC (ETP+APC) showed an excellent correlation with the APCsr (r=0.95; slope 6.6×10
–3
; 95%CI: 6.3×10
–3
to 6.9×10
–3
) indicating that the APCsr is entirely determined by the ETP
+APC
. In conclusion, OC use increases thrombin generation, but differential effects of second and third generation OCs on the protein C system likely determine the differences in the risk of venous thrombosis between these kinds of OC.
Background Hemophilia A (HA) is characterized by decreased or absent factor VIII (FVIII) activity. Current FVIII assays are based on clotting time and thus only provide information about the ...initiation of coagulation. In contrast, thrombin generation assays (TGAs) can be used to measure the full coagulation spectrum of initiation, propagation, and termination that provide information on the whole course of thrombin generation and inhibition. However, the commercially available TG kits lack sensitivity for measurements of hemophilia plasma within lower FVIII ranges, which is essential for explaining differences in bleeding phenotypes in hemophiliacs at clinically low levels of FVIII.
Aims Optimization of the TGA for measurements of low FVIII levels in severe HA patients.
Methods TGA measurements were performed in severe HA pooled plasma (n = 10). Investigations of several preanalytical and analytical variables of the assay were performed in a stepwise process and adjusted based on sensitivity toward intrinsic coagulation activation.
Results TGA initiated by tissue factor (TF) alone at varying concentrations was unable to significantly differentiate between FVIII levels below 20%. In contrast, TGA activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. In addition, a representative TGA curve at trough levels could only be produced using the dual TF/FXIa TGA.
Conclusion We propose a critical optimization for the setup of the TGA for measurements in severe HA plasma. The dual TF/FXIa TGA shows increased sensitivity, especially in lower FVIII ranges, which allows for better individual characterization at baseline, prediction of interventions, and follow-up.
Protein S expresses cofactor activity for activated protein C (APC) by enhancing the APC-catalyzed proteolysis at R306 in factor Va. It is generally accepted that only free protein S is active and ...that complex formation with C4b-binding protein (C4BP) inhibits the APC-cofactor activity of protein S. However, the present study shows that protein S-C4BP expresses APC-cofactor activity and stimulates APC-catalyzed proteolysis at R306 more than 10-fold, but instead inhibits proteolysis at R506 by APC 3- to 4-fold. Free protein S stimulates APC-catalyzed cleavage at R306 approximately 20-fold and has no effect on cleavage at R506. The resulting net effect of protein S-C4BP complex formation on APC-catalyzed factor Va inactivation is a 6- to 8-fold reduction in factor Va inactivation when compared with free protein S, which is not explained by inhibition of APC-cofactor activity of protein S at R306, but by generation of a specific inhibitor for APCcatalyzed proteolysis at R506 of factor Va. These results are of interest for carriers of the factor VLeiden mutation (R506Q), as protein S-C4BP effectively enhances APC-catalyzed factor Va (R306) inactivation in plasma containing factor VLeiden.
Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via ...formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nM). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nM) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nM). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.
Summary
Background
Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To ...identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis.
Method
We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor V
Leiden
(n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60).
Results
The thrombin generation-based APC resistance test was sensitive for the factor V
Leiden
and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin-or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases.
Interpretation
The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.
Activated protein C (APC) resistance, determined with a thrombin‐generation‐based APC resistance test, may explain risk differences of venous thrombosis in users of second‐ and third‐generation oral ...contraceptives (OC). To clinically validate this test, we analysed the Leiden thrombophilia case–control study (474 patients with a first episode of deep vein thrombosis and 474 age‐ and sex‐matched control subjects). Data for men and women were analysed separately. As hormonal status in women is known to strongly influence the APC sensitivity ratio (APCsr), additional strata (OC use and menopausal state) were defined. The APCsr was higher in all patients than in control subjects. Odds ratios (OR), using the 90th percentile of all control subjects (APCsr > 4·5) as cut‐off, were: 7·5 95% confidence interval (CI) 1·6–33·8 for men, 3·0 (95% CI 1·0–8·8) for premenopausal women not using OC, 4·8 (95% CI 1·6–14·7) for premenopausal women using OC and 4·7 (95% CI 1·4–15·6) for postmenopausal women. After excluding the carriers of factor V Leiden, the OR became infinite for men (no control had an APCsr > 4·5), 1·4 (95% CI 0·2–8·2) for premenopausal women not using OC, 3·4 (95% CI 1·1–10·8) for premenopausal women using OC and 3·6 (95% CI 0·6–20·5) for postmenopausal women. A high APCsr, determined with the thrombin‐generation‐based APC resistance test, predicts venous thrombotic risk, in populations with and without factor V Leiden. In addition, acquired APC resistance resulting from OC use predicts an increased risk for venous thrombosis independent of factor V Leiden.
Summary
The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous ...thrombosis associated with OC therapy. Here we report the results of a cyclecontrolled randomized cross-over study, in which we determined the effects of so-called second and third generation OC’s on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 µg levonorgestrel/30 µg ethinylestradiol) or a third generation OC (150 µg desogestrel/30 µg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), α
2
-macroglobulin (α
2
-M), α
1
-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of αc
2
-M, α
1
-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETPbased (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.
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