Thrombin generation‐based assays to measure the activity of the TFPI–protein S pathway in plasma from normal and protein S‐deficient individuals

Summary. Background: Protein S acts as a cofactor for full‐length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective: To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme‐linked immunosorbent assay (ELISA) to determine the level of full‐length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S‐deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti‐protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti‐TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S‐deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full‐length TFPI levels, which were significantly lower in protein S‐deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full‐length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.     

High levels of protein C are determined by PROCR haplotype 3

Summary. Background: Genetic determinants of plasma levels of protein C (PC) are poorly understood. Recently, we identified a locus on chromosome 20 determining high PC levels in a large Dutch pedigree with unexplained thrombophilia. Candidate genes in the LOD‐1 support interval included FOXA2, THBD and PROCR. Objectives: To examine these candidate genes and their influence on plasma levels of PC. Patients/Methods: Exons, promoter and 3′UTR of the candidate genes were sequenced in 12 family members with normal to high PC levels. Four haplotypes of PROCR, two SNPs in the neighboring gene EDEM2 and critical SNPs encountered during resequencing were genotyped in the family and in a large group of healthy individuals (the Leiden Thrombophilia Study (LETS) controls). Soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) plasma levels were measured in the family. Results:PROCR haplotype 3 (H3) and FOXA2 rs1055080 were associated with PC levels in the family but only PROCR H3 was also associated with plasma levels in the healthy individuals. Carriers of both variants had higher PC levels than carriers of only PROCR H3 in the family but not in healthy individuals, suggesting that a second determinant is present. EDEM2 SNPs were associated with PC levels, but their effect was small. PC and sEPCR levels were associated in both studies. sTM was not associated with variations of THBD or PC levels. Conclusions: Chromosome 20 harbors genetic determinants of PC and sEPCR levels and the analysis of candidate genes suggests that the PROCR locus is responsible.     

Paternal endothelial protein C receptor 219Gly variant as a mild and limited risk factor for deep vein thrombosis during pregnancy

Summary. Background: Half of all venous thromboembolism (VTE) cases during pregnancy are associated with a maternal thrombophilia. The influence of paternal genotype on the placenta and in the genesis of VTE has not been described. Objectives: To determine if the maternal and paternal Ser219Gly dimorphism of the endothelial protein C receptor (EPCR), evaluated through detection of the PROCR 6936G allele, is a risk factor for VTE during pregnancy. Methods: Using a case‐control study nested in the NOHA first cohort of primigravidae, 66 patient couples with a first episode of gestational VTE and randomly selected non‐thrombotic control couples were investigated. For each couple, factor V gene (F5) G1691A, factor II gene (F2) G20210A, factor XII gene (F12) C46T and PROCR A6936G polymorphisms were determined. Results: Only maternal F5 1691A, F2 20210A and F12 46T alleles were independently associated with iliac and infra‐iliac deep vein thromboses (DVT). The maternal PROCR 6936G allele was a mild risk factor for iliac DVT (OR = 5.5 [2.3–13.0]). The paternal PROCR 6936G allele was also a mild independent risk factor for iliac DVT (OR = 2.6 [1.1–6.2]) and only during pregnancy (rather than postpartum) among maternal carriers of the F5 1691A allele (OR = 77.6 [4.2 to > 999.9]). Conclusions: The paternal PROCR 6936G allele could be a risk factor for maternal iliac DVT. Its impact was milder than the F5 1691A and F2 20210A polymorphisms in mothers. We hypothesize that the prothrombotic effect of the paternal PROCR 6936G allele is localized. Therefore, DVT during pregnancy may be influenced by trophoblastic cell‐surface proteins inherited from both maternal and paternal alleles.     

The chemotherapy metabolite acrolein upregulates thrombin generation and impairs the protein C anticoagulant pathway in animal‐based and cell‐based models

Summary. Background: Thrombosis is a common complication in cancer patients receiving chemotherapy regimens that include cyclophosphamide. However, the mechanisms by which these agents increase this risk are largely uncharacterized. Objectives: To examine the effects of cyclophosphamide and its metabolite acrolein on procoagulant and anticoagulant pathways in both cell‐based and animal‐based models. Methods: Thrombin and activated protein C (APC) generation were measured in defibrinated plasma exposed to acrolein‐treated endothelial and smooth muscle cells. Tissue factor (TF) activity was measured on acrolein‐treated cells. Cell surface levels of phosphatidylserine, TF, endothelial protein C receptor and thrombomodulin were measured. Healthy BALB/c mice received injections of saline (control), acrolein, or cyclophosphamide; blood was collected, and plasma thrombin–antithrombin (TAT) complex, protein C and APC levels were analyzed. Results: Exposure of acrolein‐treated endothelial and smooth muscle cells to defibrinated plasma increased thrombin generation in the plasma. This was associated with enhanced phosphatidylserine exposure and/or increased TF activity on acrolein‐treated cells. Despite elevated levels of thrombin generation, plasma APC levels were not elevated. In vivo, treatment of mice with cyclophosphamide and acrolein resulted in elevations of plasma TAT complex levels, whereas APC levels remained low. Conclusions: This is the first study to examine thrombin generation and the APC pathway in chemotherapy‐treated mice. Cyclophosphamide and acrolein appear to upregulate procoagulant pathways, while impairing endogenous anticoagulant pathways. This may explain, in part, the increased risk of thrombosis observed in cancer patients receiving cyclophosphamide‐containing chemotherapy.     

Genome scan of clot lysis time and its association with thrombosis in a protein C‐deficient kindred

Summary. Background: Previously, we found increased clot‐lysis time (CLT), as measured with a plasma‐based assay, to increase the risk of venous thrombosis in two population‐based case–control studies. The genes influencing CLT are as yet unknown. Patients/Methods: We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis. Results: Protein C‐deficient family members had shorter CLTs than non‐deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4‐fold in non‐deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9‐fold. Combining both risk factors yielded a 27.8‐fold increased risk. The heritability of CLT was 42–52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin‐activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. Conclusion: Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13.     

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1

Summary. Background: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. Objective: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. Methods: Potential agents were generated by synthesizing specific inhibitors [6‐(Lys‐Lys‐Thr‐[homo]Arg)amino‐2‐(Lys[carbobenzoxy]‐Lys[carbobenzoxy]‐O‐benzyl)naphthalenesulfonamide] (PNASN‐1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. Results: In the activated partial thromboplastin time‐based APC resistance assay, PNASN‐1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN‐1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII‐deficient plasma (FVIII:C < 1%). The addition of PNASN‐1 to tissue factor‐triggered (5 pm ) contact pathway‐inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1–51%) increased the maximum level of thrombin generated from 78 to 162 nm , which approached that observed in blood from a healthy individual (201 nm ). PNASN‐1 also caused a 47% increase in clot weight in hemophilia A blood. Conclusions: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.     

Common hemostasis and inflammation gene variants and venous thrombosis in older adults from the Cardiovascular Health Study

Summary. Background/Objectives: Age‐related changes in blood coagulation and fibrinolysis are associated with increased risk of thrombotic events. Inherited deficiencies of coagulation proteins, such as factor V (FV) Leiden and prothrombin G20210A, explain a small fraction of venous thromboembolic disease (VTE). Additional genetic factors are likely to underlie the etiology of VTE, some of which may become manifest at older ages. Methods: We tested 290 common SNPs within 51 thrombosis and inflammation genes for association with VTE in the Cardiovascular Health Study, a large, prospective cohort of older adults followed for up to 12 years. Results: There were 184 VTE events that occurred at mean age of 78 years. TagSNPs within four genes encoding FXIII subunit A (F13A), FVII activating protease (HABP2), protease activated receptor‐1 (F2R) and the urokinase receptor (PLAUR) showed the strongest evidence for association with VTE, with each gene having a global P‐value < 0.05 and at least one tagSNP false discovery rate (FDR) q‐value < 0.05. The rs3024409 variant allele of F13A1 was associated with 1.66‐fold increased risk of VTE, while the minor alleles of HABP2 rs6585234 and rs3862019, F2R rs253061 and rs153311, and PLAUR rs344782 were each associated with lower risk of VTE (hazard ratios in the range of 0.49–0.66). Consistent with the observed protective association for VTE risk, the HABP2 rs3862019 variant allele was also associated with lower activity levels of coagulation factors FVIII, FIX, FX and plasminogen. We also confirm previously reported associations between common variants of the coagulation FII, FV, FVIII, FXI, alpha‐fibrinogen and protein C genes and risk of VTE. Conclusions: These findings suggest that several novel common coagulation gene variants may be related to risk of VTE in older adults. Further studies in older adults are needed to validate these findings and assess functional molecular mechanisms.     

Prostaglandin receptors mediate effects of substances released from ischaemic rat hearts on non‐ischaemic cardiomyocytes

Background After ischaemia and during reperfusion, rat hearts release cardiodepressive substances that are putatively cyclooxygenase‐2‐dependent. The present study analyses the mechanisms by which these substances mediate their effect downstream of cyclooxygenase‐2. Materials and methods After 10 min of global stop‐flow ischaemia, isolated rat hearts were reperfused and post‐ischaemic coronary effluent was collected over a period of 30 s. Non‐ischaemic effluent collected before ischaemia was used as a control. We investigated the effect of the effluents on cell shortening and Ca⁺⁺‐metabolism, by application of fluorescence microscopy of field‐stimulated adult rat cardiomyocytes incubated with fura‐2. Cells were pre‐incubated with inhibitors of protein kinase A and C and with antagonists of protein kinase A‐dependent prostaglandin receptors. We examined the expression of prostaglandin receptors in cardiomyocytes by Western blotting. Results In contrast to non‐ischaemic effluent, post‐ischaemic effluent induced reduction of Ca⁺⁺ transient and cell shortening in the cardiomyocytes. In contrast to protein kinase C inhibitor Myr‐PKC [19–27], the protein kinase A inhibitor Rp‐cAMPS completely blocked the effect of post‐ischaemic effluent. Furthermore, we determined a cyclic adenosine monophosphate increase in cardiomyocytes that were pre‐incubated with post‐ischaemic effluent. The antagonist of prostaglandin E‐receptor EP2 AH6809 and the antagonist of receptor subtype EP4 AH23848 attenuated the effect of post‐ischaemic effluent in contrast to other antagonists of prostaglandin D and I receptors, which did not influence the effect. In lysates of adherend cardiomyocytes, expression of prostaglandin D, E and I receptors was detected by Western blotting. Conclusions The effect of post‐ischaemic effluent is mediated by the protein kinase A‐dependent prostaglandin‐receptor subtypes EP2 and EP4 downstream of cyclooxygenase‐2.     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

Thrombin upregulates the angiopoietin–Tie2 Axis: endothelial protein C receptor occupancy prevents the thrombin mobilization of angiopoietin 2 and P‐selectin from Weibel–Palade bodies

Summary. Background: Activated protein C (APC) in complex with endothelial protein C receptor (EPCR) can reverse the barrier‐disruptive and cytotoxic effects of proinflammatory cytokines by cleaving protease‐activated receptor 1 (PAR‐1). Recently, it was reported that the PAR‐1‐dependent vascular barrier‐protective effect of APC is mediated through transactivation of the angiopoietin (Ang)–Tie2 signaling pathway. The antagonist of this pathway, Ang2, is stored in Weibel–Palade bodies within endothelial cells. Objectives: To determine whether the occupancy of EPCR by its ligand can switch the PAR‐1‐dependent signaling specificity of thrombin through the Ang–Tie2 axis. Methods: We activated endothelial cells with thrombin before and after treating them with the catalytically inactive Ser195→Ala substitution mutant of protein C. The expression levels of Ang1, Ang2 and Tie2 in response to thrombin were measured by both an enzyme‐linked immunosorbent assay and a cell permeability assay in the absence and presence of small interfering RNA and a blocking antibody to Tie2. Results: Thrombin upregulated the expression of both Ang1 and Tie2 but downregulated the expression of Ang2 when EPCR was occupied by its ligand. The Ang1–Tie2‐dependent protective effect of thrombin was initiated through protein C inhibiting the rapid mobilization of Ang2 from Weibel–Palade bodies. Interestingly, the protein C mutant also inhibited the thrombin mobilization of P‐selectin. Conclusions: These results suggest a physiologic role for the low concentration of thrombin in maintaining the integrity of the EPCR‐containing vasculature through the PAR‐1‐dependent inhibition of Ang2 and P‐selectin release from Weibel–Palade bodies.     

Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment

Summary. Background: Vitamin K 2,3‐epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. Objectives: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. Patients/Methods: Six hundred and twenty‐six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. Results: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26→Thr, His28→Gln, Asp36→Gly, Ser52→Trp, Ser56→Phe, Trp59→Leu, Trp59→Cys, Val66→Gly, Gly71→Ala, Asn77→Ser, Asn77→Tyr, Ile123→Asn, and Tyr139→His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29→Leu, Asp36→Tyr, and Val66→Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non‐VKORC1*2 and wild‐type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane–lumen interface. Conclusions: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.     

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels

Summary. Objectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain‐of‐function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. Methods and Results: We genotyped the control populations of two population‐based studies for four AVPR2 variations: a‐245c, G12E, L309L, and S331S. Rare alleles of a‐245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G‐V2R or 12E‐V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three‐fold in 12E‐V2R–green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (K_D) was 4.5 nm [95% confidence interval (CI) 3.6–5.4] for 12E‐V2R–GFP and 16.5 nm (95% CI 10.1–22.9) for 12G‐V2R–GFP. AVP‐induced cAMP generation was enhanced in 12E‐V2R–GFP cells. Conclusions: The 12E‐V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVIII.     

Extracellular histones increase plasma thrombin generation by impairing thrombomodulin‐dependent protein C activation

See also Borissoff JI, ten Cate H. From neutrophil extracellular traps release to thrombosis: an overshooting host‐defense mechanism? This issue, pp 1791–4. Summary. Background: Histones are basic proteins that contribute to cell injury and tissue damage when released into the extracellular space. They have been attributed a prothrombotic activity, because their injection into mice induces diffuse microvascular thrombosis. The protein C–thrombomodulin (TM) system is a fundamental regulator of coagulation, particularly in the microvasculature, and its activity can be differentially influenced by interaction with several cationic proteins. Objective: To evaluate the effect of histones on the protein C–TM system in a plasma thrombin generation assay and in purified systems. Methods: The effect of histones on plasma thrombin generation in the presence or absence of TM was analyzed by calibrated automated thrombinography. Protein C activation in purified systems was evaluated by chromogenic substrate cleavage. The binding of TM and protein C to histones was evaluated by solid‐phase binding assay. Results: Histones dose‐dependently increased plasma thrombin generation in the presence of TM, independently of its chondroitin sulfate moiety. This effect was not caused by inhibition of activated protein C activity, but by the impairment of TM‐mediated protein C activation. Histones were able to bind to both protein C and TM, but the carboxyglutamic acid domain of protein C was required for their effect. Histones H4 and H3 displayed the highest activity. Importantly, unlike heparin, DNA did not inhibit the potentiating effect of histones on thrombin generation. Conclusions: Histones enhance plasma thrombin generation by reducing TM‐dependent protein C activation. This mechanism might contribute to microvascular thrombosis induced by histones in vivo at sites of organ failure or severe inflammation.     

Association of coagulation‐related and inflammation‐related genes and factor VIIc levels with stroke: the Cardiovascular Health Study

Summary. Background: Thrombosis and inflammation are critical in stroke etiology, but associations of coagulation and inflammation gene variants with stroke, and particularly factor VII levels, are inconclusive. Objectives: To test the associations between 736 single‐nucleotide polymorphisms (SNPs) between tagging haplotype patterns of 130 coagulation and inflammation genes, and stroke events, in the 5888 participants aged ≥ 65 years of the observational Cardiovascular Health Study cohort. Patients/Methods: With 16 years of follow‐up, age‐adjusted and sex‐adjusted Cox models were used to estimate associations of SNPs and FVIIc levels with future stroke. Results: Eight hundred and fifteen strokes occurred in 5255 genotyped participants without baseline stroke (748 ischemic strokes; 586 among whites). Among whites, six SNPs were associated with stroke, with a nominal P‐value of < 0.01: rs6046 and rs3093261 (F7); rs4918851 and rs3781387 (HABP2); and rs3138055 (NFKB1A) and rs4648004 (NFKB1). Two of these SNPs were associated with FVIIc levels (units of percentage activity): rs6046 (β = ? 18.5, P = 2.38 × 10^?83) and rs3093261 (β = 2.99, P = 3.93 × 10^?6). After adjustment for age, sex, race, and cardiovascular risk factors, the association of FVIIc quintiles (Q) with stroke were as follows (hazard ratio; 95% confidence interval): Q1, reference; Q2, 1.4, 1.1–1.9); Q3, 1.1, 0.8–1.5); Q4, 1.5, 1.1–2.0); and Q5, 1.6, 1.2–2.2). Associations between SNPs and stroke were independent of FVIIc levels. Conclusions:   Variations in FVII‐related genes and FVIIc levels were associated with risk of incident ischemic stroke in this elderly cohort, suggesting a potential causal role for FVII in stroke etiology.     

PROC c.574_576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population

See also Hamasaki N. Unmasking Asian thrombophilia: is APC dysfunction the real culprit? This issue, pp 2016–8. Summary. Background: There are ethnic differences in the genetic risk factors for venous thrombosis (VT). The genetic causes of VT in the Chinese population are not fully understood. Objectives: To identify possible common abnormal factors that could contribute to thrombosis susceptibility. Methods/Results: We measured the levels of nine types of plasma coagulation factor, three types of anticoagulation factor and two types of fibrinolytic factor in 310 VT patients. Factor V activity was higher in 32 cases. Eleven of the 32 cases also had low protein C (PC) or protein S (PS) activities, indicating PC or PS deficiency. No other abnormalities were observed in the other 21 cases. All of the samples were sensitive to activated PC inactivation. Therefore, the abnormal factor involved may be FV inactivator or its cofactor rather than FV itself. Resequencing identified a common PROC c.574_576del variant in 10 of the 32 subjects. In a case–control study, this variant was detected in 68 of the 1003 patients and in 25 of the 1031 controls. It had an adjusted odds ratio of 2.71 (95% confidence interval [CI] 1.68–4.36). PC amidolytic activities of most variant carriers were similar to those of non‐carriers, but the mean anticoagulant activity was only 72.7 U dL^?1. Expression studies in vitro showed that the anticoagulant activity of the mutant PC was 43.6% of that of the wild‐type PC. Conclusions: We identified what is, so far, the most common genetic risk factor for VT in the Chinese population, with its prevalence being approximately 2.36%.     

Activated protein C modulates inflammation,apoptosis and tissue factor procoagulant activity by regulating endoplasmic reticulum calcium depletion in blood monocytes

Summary. Background: The endoplasmic reticulum (ER) is responsible for the synthesis and folding of secretory, transmembrane and ER‐resident proteins. Conditions that impair protein folding or overwhelm its protein folding capacity disrupt ER homeostasis, thereby causing ER stress. ER stress‐induced apoptosis and inflammation are involved in the pathogenesis of inflammatory diseases. Activated protein C (APC) inhibits inflammation and apoptosis in monocytes, and this may partly explain the protective effects of APC treatment in severe sepsis. However, the precise molecular pathways by which APC modulates these effects remain unknown. Objectives: To investigate whether APC modulates the ER stress response in human monocytes. Methods: We treated monocytes with ER stress‐inducing agents in the presence or absence of APC to determine the effect on this response. Protein and mRNA levels were determined by immunoblotting and real‐time PCR, respectively. Enzyme assays and flow cytometry were used to determine the role of APC in this model. Results: In thapsigargin (Tg)‐treated cells, APC dampened unfolded protein response activation, as indicated by reduced levels of the 78‐kDa glucose‐regulated protein (GRP78), in an endothelial protein C receptor‐independent and protease‐activated receptor‐1‐independent manner. Consistent with this, APC decreased phosphorylated eukaryotic translational initiation factor 2α and C/EBP homologous protein levels induced by Tg. APC inhibited Tg‐induced ER Ca²⁺ flux and reactive oxygen species generation. Functionally, APC diminished Tg‐induced caspase‐3 activity and degradation of the nuclear factor kappaB inhibitor IκBα. Furthermore, APC dampened the induction of tissue factor procoagulant activity facilitated by Tg. Conclusions: These studies suggest that APC modulates the adverse effects of ER Ca²⁺ depletion in human monocytes.     

Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro

To cite this article: Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9 : 1608–19. Summary. Background: Protease‐activated receptors (PARs) are G‐protein‐coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR‐agonist peptides (PAR‐APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin‐enriched membrane microdomains in which PAR1 and caveolin‐1 associate. Objectives: The aim of this study was to determine the effects of cholesterol‐lowering agents (statins) and cholesterol‐loading lipoprotein on PAR1‐AP‐mediated and PAR2‐AP‐mediated TF induction in HUVECs. Results: Statins completely prevented TF induction by PAR‐APs in an isoprenoid‐independent manner, induced the delocalization of PAR1 from caveolin‐enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR‐AP‐mediated extracellular signal‐related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain‐associated G‐protein α, caveolin‐1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1‐AP‐induced TF, probably as a result of the increased abundance of PAR1 and the Src and G‐protein α signaling molecules in the caveolin‐1‐enriched fraction, without affecting PAR1 mRNA. Conclusions: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR‐APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.     

Prevalence,biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one‐stage and chromogenic factor VIII activity

Summary. Background: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one‐stage assay. However, comparisons of these results with those of two‐stage assays can reveal discrepancies and suggest misdiagnosis. Patients/Methods: In this monocentric study, we measured FVIII:C with two methods (one‐stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. Results: Thirty‐six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. Conclusions: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of ‘inverse’ discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure–function relationships.