Biomedical subjects

Jan Rosing

Publications and source records attributed to Jan Rosing.

35 records · Page 2Linked to original sources

PubMed2004

Effect of raloxifene on activated protein C (APC) resistance in postmenopausal women and on APC resistance and homocysteine levels in elderly men: two randomized placebo-controlled studies.

Raloxifene, a selective estrogen receptor modulator, like hormonal replacement therapy increases the risk of venous thromboembolism in postmenopausal women. A possible explanation for the increased thrombotic risk could be an increase in acquired resistance to activated protein C (APC). In two randomized, placebo-controlled, double-blind studies we determined the effect of raloxifene on the normalized APC sensitivity ratios (nAPCsr). The nAPCsr were determined with the thrombin generation-based APC resistance test. In the first study 83 postmenopausal women (age, 51.1 +/- 2.7 years) randomly received daily 0.625 mg conjugated equine estrogen and 2.5 mg medroxyprogesterone acetate (n=17), 60 mg raloxifene (n=23), 150 mg raloxifene (n=20) or placebo (n=23) for 24 months. At baseline and after 6, 12 and 24 months the nAPCsr were measured. In the second study 30 elderly men (age, 64.4 +/- 2.4 years) randomly received 120 mg raloxifene (n=15) or placebo (n=15) for 3 months. At baseline and after 3 months the nAPCsr and fasting homocysteine levels were measured. In postmenopausal women conjugated equine estrogen/medroxyprogesterone acetate significantly increased the nAPCsr from 1.26 +/- 0.82 to 2.87 +/- 0.86 at 24 months (P <0.0005 compared with placebo). Raloxifene had no significant effect on nAPCsr compared with placebo in both women and men. The results did not change after excluding carriers of factor V Leiden. Also fasting homocysteine levels were not affected by raloxifene in the aging men. It is concluded that raloxifene, in contrast to combined hormonal replacement therapy, does not increase APC resistance.

Activated Protein C Resistance↗

PubMed2004

Factor V Leiden: a disorder of factor V anticoagulant function.

PURPOSE OF REVIEW: Activated protein C (APC) resistance, which is often associated with the factor V R506Q (FV Leiden) mutation, is a common risk factor for venous thrombosis. Study of the mechanism of APC resistance has revealed that coagulation FV stimulates the APC-catalysed inactivation of FVIIIa, and that this anticoagulant function of FV is impaired in FV Leiden. The present review covers the discovery, the physiological significance and the structural requirements of the APC-cofactor activity of FV. RECENT FINDINGS: Recent in vitro and in vivo experiments indicate that the anticoagulant activity of FV is physiologically relevant and that FV plays a major role in the maintenance of the haemostatic balance. Quantitative and functional defects of the APC-cofactor activity of FV lead to increased thrombin generation and are associated with a prothrombotic state. Although the structural requirements for the expression of the APC-cofactor activity of FV are now beginning to be unravelled, the underlying molecular mechanism remains elusive. SUMMARY: The APC-cofactor activity of FV and its impairment in FV Leiden can explain the different thrombosis risks associated with heterozygosity, homozygosity and pseudo-homozygosity for FV Leiden. Elucidation of the molecular mechanism of the anticoagulant function of factor V may provide novel targets for the design of antithrombotic drugs.

Activated Protein C Resistance↗

PubMed2004

Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.

Blotting, Western↗

PubMed2004

The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities.

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

Anticoagulants↗

PubMed2003

Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679.

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.

Animals↗

PubMed2003

Effect of second- and third-generation oral contraceptives on the protein C system in the absence or presence of the factor VLeiden mutation: a randomized trial.

A plausible mechanism to explain thrombotic risk differences associated with the use of second- and third-generation oral contraceptives (OCs), particularly in carriers of factor V(Leiden), is still lacking. In a double-blind trial, 51 women without and 35 women with factor V(Leiden) were randomized to either a second- (30 microg ethinylestradiol/150 microg levonorgestrel) or third- (30 microg ethinylestradiol/150 microg desogestrel) generation OC. After 2 cycles of use and a wash-out of 2 cycles, the participants continued with the corresponding progestagen-only preparation. Hemostatic variables that probe the activity of the anticoagulant protein C system were determined. Compared with levonorgestrel, desogestrel-containing OCs significantly decreased protein S and increased activated protein C (APC) resistance in both groups. OCs with desogestrel had the most pronounced effects in carriers of factor V(Leiden). Progestagen-only preparations caused changes of anticoagulant parameters opposite to those of combined OCs, which in a number of cases were more pronounced with levonorgestrel. Our data show that progestagens in combined OCs counteract the thrombotic effect of the estrogen component. The higher thrombotic risk associated with third-generation OCs compared with second-generation OCs may be explained by the fact that desogestrel appeared less antithrombotic than levonorgestrel, especially in women with factor V(Leiden).

Adolescent↗

PubMed2003

Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation.

We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency.

Adult↗

PubMed2003

Effect of oral and transdermal estrogen replacement therapy on hemostatic variables associated with venous thrombosis: a randomized, placebo-controlled study in postmenopausal women.

OBJECTIVE: The purpose of this study was to investigate whether the effect of transdermal estrogen therapy in postmenopausal women differs from that of oral therapy with regard to resistance to activated protein C (APC), an important risk factor for venous thrombosis, and levels of related proteins, such as protein S, protein C, and prothrombin. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled study, 152 healthy hysterectomized postmenopausal women received daily either placebo (n=49), transdermal 17beta-estradiol (E2) 50 microg (tE2 group, n=33), oral E2 1 mg (oE2 group, n=37), or oral E2 1 mg combined with gestodene 25 microg (oE2+G group, n=33) for 13 28-day treatment cycles, followed by 4 cycles of placebo for each group. Plasma samples were collected at baseline and in cycles 4, 13, and 17. In cycle 13, significant increases versus baseline and placebo were found in normalized APC sensitivity ratios (nAPCsr) in all treated groups (tE2, +26.9%; oE2, +102.7%; oE2+G, +69.9%). Increases in nAPCsr were significantly higher in the oral treatment groups than in the tE2 group. In addition, compared with baseline and placebo, after 13 cycles, decreases were observed in total protein S (tE2, -4.1%; oE2, -7.9%; oE2+G, -5.8%), free protein S (tE2, -7.1%; oE2, -8.4%; oE2+G, -5.2%), and protein C in the oE2+G group (-6.4%), but these changes did not explain the increase in nAPCsr. Changes in prothrombin were small and also did not affect the nAPCsr. CONCLUSIONS: Increases were observed in resistance to APC, which were more pronounced in the oral treatment groups than in the transdermal group. The increase in resistance to APC was not explained by changes in protein S, protein C, or prothrombin and may contribute to the increased incidence of venous thrombosis in users of hormone replacement therapy.

Activated Protein C Resistance↗

PubMed2003

The APC-independent anticoagulant activity of protein S in plasma is decreased by elevated prothrombin levels due to the prothrombin G20210A mutation.

Protein S exhibits anticoagulant activity independent of activated protein C (APC). An automated factor Xa-based one-stage clotting assay was developed that enables quantification of the APC-independent activity of protein S in plasma from the ratio of clotting times (protein S ratio [pSR]) determined in the absence and presence of neutralizing antibodies against protein S. The pSR was 1.62 +/- 0.16 (mean +/- SD) in a healthy population (n = 60), independent of plasma levels of factors V, VIII, IX, and X; protein C; and antithrombin, and not affected by the presence of factor V Leiden. The pSR strongly correlates with the plasma level of protein S and is modulated by the plasma prothrombin concentration. In a group of 16 heterozygous protein S-deficient patients, the observed mean pSR (1.31 +/- 0.09) was significantly lower than the mean pSR of the healthy population, as was the pSR of plasma from carriers of the prothrombin G20210A mutation (1.47 +/- 0.21; n = 46). We propose that the decreased APC-independent anticoagulant activity of protein S in plasma with elevated prothrombin levels may contribute to the thrombotic risk associated with the prothrombin G20210A mutation.

Antibodies↗

PubMed2003

Acquired resistance to activated protein C in breast cancer patients.

In 56 women with a lymph-node-positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC-sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC-sr 2.02 vs 1.03 in the healthy control subjects (P < 0.001). No difference in APC-sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108.0%vs 96.0% and 94.5% (P = 0.008 and P = 0.007). The APC-sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue-factor-based APC resistance test might contribute to the cancer-related hypercoagulability.

Activated Protein C Resistance↗

PubMed2003

Activated protein C resistance determined with a thrombin generation-based test predicts for venous thrombosis in men and women.

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.

Activated Protein C Resistance↗

PubMed2003

Changes of hemostatic variables during oral contraceptive use.

The use of oral contraceptives (OCs) has been known for many years to affect significantly almost all hemostatic parameters, but the challenge to relate these changes in a meaningful way to OC-induced increased venous thrombotic risk has not been met. New insights indicate that at least part of the answer can be found in the net effect of OC use on the efficacy with which the protein C pathway down-regulates thrombin formation. During OC use the (blood) plasma of a woman becomes resistant to the anticoagulant action of activated protein C (APC). The extent of this so-called acquired APC resistance as determined in a thrombin generation-based assay correlates remarkably well with the risk increases observed in clinical studies. Recent evidence indicates that the prothrombotic effect of the estrogen component ethinylestradiol in combined OC is counteracted by the progestagen component present in these preparations and that third-generation progestagens such as desogestrel or gestodene are less efficient with respect to this than the second-generation progestagen levonorgestrel.

Activated Protein C Resistance↗

PubMed2003

Endogenous factor V synthesis in megakaryocytes contributes negligibly to the platelet factor V pool.

BACKGROUND AND OBJECTIVES: Coagulation factor V (FV) is distributed between two pools: 80% circulates in plasma and 20% is stored in platelets. The aim of the study was to determine the origin of platelet FV. DESIGN AND METHODS: We investigated a FV Leiden heterozygous patient who had received an allogeneic bone marrow transplant from a normal donor. The patient had been referred to our laboratory for his marked activated protein C (APC) resistance in the apparent absence of FV Leiden. Analysis of the DNA from a buccal swab showed that the patient was indeed a heterozygous carrier of FV Leiden. The difference in FV genotype between the hepatocytes (heterozygous FV Leiden) and the blood cells (homozygous normal) of the patient provided a good model to investigate the origin of platelet FV. Platelets were isolated from the patient and the bone marrow donor and activated with thrombin and ionomycin to release and activate FV. APC was then added and the inactivation of platelet FVa was followed over time with a highly sensitive prothrombinase-based assay. RESULTS: While the donor's platelet FVa showed a normal inactivation time course, the patient's platelet FVa was considerably resistant to APC. The kinetic pattern of APC-catalyzed inactivation of the patient's platelet FVa was indistinguishable from that of plasma FVa from a FV Leiden heterozygote. INTERPRETATION AND CONCLUSIONS: These data indicate that platelet FV is derived from plasma and that endogenous FV synthesis by megakaryocytes contributes negligibly to the platelet FV pool.

Activated Protein C Resistance↗

PubMed2002

Increased resistance to activated protein C after short-term oral hormone replacement therapy in healthy post-menopausal women.

As hormone replacement therapy is associated with an early excess risk of venous thrombosis, we investigated the effect of different oral hormone replacement therapies on resistance to activated protein C, and on levels of factor VIII antigen (FVIII:Ag) and factor XI antigen (FXI:Ag). In a prospective, randomized, placebo-controlled 12-week study, 60 healthy post-menopausal women daily received either placebo (n = 16) or 2 mg of micronized 17beta-oestradiol, either alone (E2, n = 16) or sequentially combined with dydrogesterone 10 mg (E2 + D, n = 14) or trimegestone 0.5 mg (E2 + T, n = 14). Medication was given orally. Normalized activated protein C sensitivity ratios (nAPCsr) were determined by quantifying the effect of activated protein C on the endogenous thrombin potential. FVIII:Ag and FXI:Ag were determined by enzyme-linked immunosorbent assay. Compared with baseline and placebo, the nAPCsr increased (92% to 142%; all P < 0.001) in all active treatment groups after both 4 and 12 weeks. Compared with placebo, hormone replacement therapy was not associated with significant changes in FVIII:Ag. After 4 and 12 weeks, FXI:Ag levels were significantly decreased in the E2 group (mean percentage changes from baseline versus placebo: -15.0%, P = 0.001 at 4 weeks and -16.6%, P = 0.003 at 12 weeks) and in the E2 + D group (-10.4%, P = 0.02 and -10.4%, P = 0.02). In conclusion, all hormone replacement regimens were associated with a large increase in resistance to activated protein C. In contrast, hormone replacement therapy had no effect on FVIII:Ag. Oral E2 and E2 + D had a small, favourable effect on FXI:Ag.

Activated Protein C Resistance↗

PubMed2002

Effects of (pre-)analytical variables on activated protein C resistance determined via a thrombin generation-based assay.

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APC-resistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (-20 degrees C vs. -80 degrees C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Activated Protein C Resistance↗

PubMed2002

Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test.

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 Resistance↗

PubMed2002

Reduced factor V concentration and altered FV1/FV2 ratio do not fully explain R2-associated APC-resistance.

Carriership of the factor V (FV) R2 haplotype is associated with mild APC-resistance, moderately reduced FV levels and a relative increase of the more thrombogenic FV isoform, FV1. Since low FV levels and increased FV1 can theoretically cause APC-resistance, we investigated whether these alterations can quantitatively account for R2-associated APC-resistance. In order to determine the effect of FV concentration and FV isoform composition on the APC-response, we reconstituted FV-deficient plasma with purified FV1 and FV2 in different molar ratios and to varying FV concentrations. APC sensitivity ratios (APCsr) were determined with the Coatest APC Resistance V, which probes the effect of APC on both FVa- and FVIIIa-inactivation, and with the Immunochrom APC response test, which only quantifies the effect of APC on FVIII(a)-inactivation. In both assays, low FV concentrations and/or high relative amounts of FV1 rendered plasma samples more resistant to APC. APCsr were also determined in FV-deficient plasma reconstituted with purified FV at levels and isoform ratios observed in R2-homozygotes (98% FV, 42% FV1) and age-matched controls (119% FV, 26% FV1). In both tests the APCsr of reconstituted control plasma was the same as that of plasma from controls, whereas reconstituted R2-plasma was less APC-resistant than plasma from homozygous carriers of the R2 haplotype. We conclude that the low FV levels and altered FV isoform ratio cannot fully explain R2-associated APC-resistance.

Activated Protein C Resistance↗