Tissue factor-bearing microparticles and CA19.9: two players in pancreatic cancer-associated thrombosis?

BACKGROUND: Cancer-related venous thromboembolism (VTE) heralds a poor prognosis, especially in pancreatic adenocarcinoma (PAC). Tissue factor (TF) is implicated as one of the main culprits in PAC-associated VTE and disease progression. METHODS: In a prospective cohort study of 79 PAC patients, we measured plasma CA19-9 and microparticle-associated TF activity (MP-TF activity). In addition, we enumerated TF(+)MPs and MUC1(+)MPs in plasma (n=55), and studied the expression of TF, MUC1, CD31 and CD68 in tumour tissue (n=44). RESULTS: Plasma MP-TF activity was markedly elevated in PAC patients with VTE compared with those without (median: 1925 vs 113 fM Xa min(-1); P<0.001) and correlated with the extent of thromboembolic events, metastatic disease and short survival. Similar results were found for CA19-9. Patients with massively progressing thrombosis and cerebral embolisms despite anticoagulant therapy (n=3) had the highest MP-TF activities (12 118-40 188 fM Xa min(-1)) and CA19-9 (40 730-197 000 kU l(-1)). All tumours expressed MUC1 and TF. MP-TF activity did not correlate with intensity of TF expression in adenocarcinoma cells, but corresponded with numbers of TF(+) macrophages in the surrounding stroma. CONCLUSIONS: Circulating TF(+)MPs and mucins may concertedly aggravate coagulopathy in PAC. Understanding of underlying mechanisms may result in new treatment strategies for VTE prevention and improvement of survival.

Fibrinogen γ' increases the sensitivity to activated protein C in normal and factor V Leiden plasma.

Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance.

Association of the thrombomodulin gene c.1418C>T polymorphism with thrombomodulin levels and with venous thrombosis risk.

OBJECTIVE: To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. APPROACH AND RESULTS: The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. CONCLUSIONS: The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane-bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

Anti-plasminogen antibodies compromise fibrinolysis and associate with renal histology in ANCA-associated vasculitis.

Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA. Eighteen of 74 (24%) UK and 10/38 (26%) Dutch patients with AAV had anti-plasminogen antibodies compared with 0/50 and 1/61 (2%) of controls. We detected anti-plasminogen antibodies in both PR3-ANCA- and MPO-ANCA-positive patients. In addition, we identified anti-tissue plasminogen activator (tPA) antibodies in 13/74 (18%) patients, and these antibodies were more common among patients with anti-plasminogen antibodies (P = 0.011). Eighteen of 74 AAV-IgG (but no control IgG) retarded fibrinolysis in vitro, and this associated with anti-plasminogen and/or anti-tPA antibody positivity. Only 4/18 AAV-IgG retarded fibrinolysis without harboring these antibodies; dual-positive samples retarded fibrinolysis to the greatest extent. Patients with anti-plasminogen antibodies had significantly higher percentages of glomeruli with fibrinoid necrosis (P < 0.05) and cellular crescents (P < 0.001) and had more severely reduced renal function than patients without these antibodies. In conclusion, anti-plasminogen and anti-tPA antibodies occur in AAV and associate with functional inhibition of fibrinolysis in vitro. Seropositivity for anti-plasminogen antibodies correlates with hallmark renal histologic lesions and reduced renal function. Conceivably, therapies that enhance fibrinolysis might benefit a subset of AAV patients.

Fibrinogen gamma gene 3'-end polymorphisms and risk of venous thromboembolism in the African-American and Caucasian population.

Genetic determinants of venous thromboembolism (VTE) in the African-American population are poorly characterised. It was recently shown that fibrinogen gamma gene (FGG) polymorphisms 10034C>T and 9340T>C influence VTE risk in the Caucasian population. In the African-American population these polymorphisms are common, with allele frequencies above 25%. Here we evaluated whether these and other FGG 3'-end polymorphisms were associated with VTE risk in the African-American population and aimed to replicate the association in the Caucasian population. We examined 557 Caucasian patients and 678 Caucasian controls, and 537 African-American patients and 586 African-American controls from the ;Genetic Attributes and Thrombosis Epidemiology' (GATE) study. In the African-American population, 10034C>T and 9340T>C marginally influenced VTE-risk, with a 20% increase in risk for 10034TT carriers and a 20% reduction in risk for 9340CC carriers. In the Caucasian population, 10034TT was associated with a 1.7-fold increase in risk, which increased to 2.1-fold for idiopathic VTE patients. 9340CC significantly reduced VTE risk approximately two-fold. In conclusion, both FGG polymorphisms 10034C>T and 9340T>C influence VTE-risk, with the strongest effects observed in the Caucasian population, confirming previous data on these polymorphisms in this population.

The role of procoagulants and anticoagulants in the development of venous thromboembolism.

Procoagulant and anticoagulant reactions play an important role in the regulation of thrombin formation during secondary hemostasis. Three phases can be recognized in the kinetics of thrombin formation: an initiation phase, a propagation phase and a termination phase. Dysregulation of thrombin formation during each of these phases by (hereditary) changes in the plasma concentration of pro- and anticoagulants contributes to the development of venous thrombosis. Most important seems the defective down-regulation of the prothrombinase activity during the termination phase. Procoagulant and anticoagulant proteins have important roles in the regulation of fibrin formation during secondary hemostasis. Under normal physiological conditions there is a delicate balance between the procoagulant and anticoagulant reactions. After damage to the vessel wall sufficient fibrin is formed to arrest bleeding and allow repair of the lesion without obstructing blood circulation. Venous thrombosis can be considered as a hemostatic process getting out of control, where massive fibrin formation has resulted in the formation of an obstructive thrombus. Such thrombus formation is believed to be facilitated by changes in the vessel wall, blood flow and the composition of the blood. During the past 50 years substantial progress has been made in our understanding of the enzymatic reactions involved in the hemostatic process. At the same time information has been obtained on particular changes in the composition of the blood which contribute to the development of venous thrombosis. Most of these changes concern the procoagulant and anticoagulant systems. In this paper I will briefly discuss how fibrin formation is regulated by procoagulant and anticoagulant reactions and how certain changes in these pathways contribute to the development of venous thrombosis.

Fibrinogen gamma' in ischemic stroke: a case-control study.

BACKGROUND AND PURPOSE: To determine the contribution of fibrinogen gamma' levels and FGG haplotypes to ischemic stroke. METHODS: Associations between fibrinogen gamma' levels, fibrinogen gamma'/total fibrinogen ratio, and FGG haplotypes with the risk of ischemic stroke were determined in 124 cases and 125 controls. RESULTS: Fibrinogen gamma'/total fibrinogen ratio was higher in patients than in controls during the acute phase of the stroke and lower in the convalescent phase 3 months after the stroke. FGG haplotype 3 (H3) was associated with a reduced risk of ischemic stroke (odds ratio 0.60; 95% CI, 0.38 to 0.94), but not with the fibrinogen gamma'/total fibrinogen ratio. In contrast, FGG-H2 was associated with a decreased fibrinogen gamma'/total fibrinogen ratio, but not with risk of stroke. CONCLUSIONS: Fibrinogen gamma'/total fibrinogen ratio is associated with ischemic stroke, especially in the acute phase of the disease. In addition, FGG-H3 haplotype appears to be protective against ischemic stroke.

Haplotypes of the EPCR gene, prothrombin levels, and the risk of venous thrombosis in carriers of the prothrombin G20210A mutation.

BACKGROUND: Haplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele. DESIGN AND METHODS: We genotyped 4678G/C and 4600A/G in 246 20210A carriers: 84 venous thromboembolism propositi and 162 relatives (13 symptomatic), and in 140 relatives not carrying the 20210A variant. Prothrombin and soluble EPCR (sEPCR) levels were also measured. RESULTS: Among propositi, the mean age at first onset was lower in carriers (35 +/- 8 years) than non-carriers of the 4600G allele (44 +/- 14 years) (p = 0.004). The probability of being free of thrombosis at age 40 was lower in 20210A carriers with the EPCR 4600G allele (p = 0.015). The frequency of the 4600G allele (p=0.002) and the levels of prothrombin antigen (p = 0.002) and sEPCR (p < 0.001) were higher in the propositi than in their asymptomatic relatives. Multivariate analyses showed that the presence of the 4600G allele (OR = 2.5, 95% confidence interval 1.3-5.0), sEPCR > 147 ng/mL (2.8, 1.5-5.2) and prothrombin > 129% (3.8, 1.8-8.3) all increased the thrombotic risk. In bivariate analysis, including the 4600G allele and sEPCR > 147 ng/mL, only the latter remained associated with risk. CONCLUSIONS: These results show that in 20210A carriers the venous thromboembolism risk is influenced both by the actual prothrombin levels and by the EPCR A3 haplotype, via its effect on sEPCR levels.

Endothelial protein C receptor polymorphisms and risk of myocardial infarction.

BACKGROUND: Haplotypes A1 and A3 in the endothelial protein C receptor gene are tagged by the 4678G/C and 4600A/G polymorphisms, respectively, and have been reported to influence the risk of venous thromboembolism. We assessed whether these haplotypes modify the risk of premature myocardial infarction. DESIGN AND METHODS: We genotyped these polymorphisms in 689 patients with premature myocardial infarction and 697 control subjects. Activated protein C and soluble endothelial protein C receptor levels were also measured. RESULTS: After adjustment for other cardiovascular risk factors, A1 and A3 haplotypes protected against premature myocardial infarction (odds ratio 0.7, 95% CI 0.4-0.8, p=0.044 and 0.5, 0.3-0.6, p<0.001, respectively). Moreover, the protective role of these haplotypes seemed to be additive, as carriers of both the A1 and A3 haplotypes had adjusted odds ratios of 0.3 (0.2-0.5, p<0.001) and 0.4 (0.2-0.8, p=0.006) compared to those carrying only the A1 or A3 haplotype, respectively. The presence of the A1 haplotype was associated with increased levels of activated protein C whereas individuals carrying the A3 haplotype showed the highest soluble endothelial protein C receptor levels. CONCLUSIONS: These results show that A1 haplotype carriers have a reduced risk of premature myocardial infarction via the association of this haplotype with increased activated protein C plasma levels. The study also shows that carriers of the A3 haplotype have a reduced risk of myocardial infarction, only in part due to increased soluble endothelial protein C levels.

Haplotypes of IL1B, IL1RN, IL1R1, and IL1R2 and the risk of venous thrombosis.

OBJECTIVE: It has been suggested that the overall effect of the major proinflammatory cytokine interleukin-1 (IL-1) on coagulation and fibrinolysis is prothrombotic. The aim of this study was to investigate whether common variations in IL1B, IL1RN, IL1R1, and IL1R2 influence the risk of venous thrombosis. METHODS AND RESULTS: In a case-control study on the causes of deep venous thrombosis, the Leiden Thrombophilia Study (LETS), we genotyped 18 single nucleotide polymorphisms (SNPs) in IL1B, IL1RN, IL1R1, and IL1R2, enabling us to tag a total of 25 haplotype groups. Overall testing of the haplotype frequency distribution in patients and controls indicated that a recessive effect was present in IL1RN (P=0.031). Subsequently the risk of venous thrombosis was calculated for each haplotype of IL1RN. Increased thrombotic risk was found for homozygous carriers of haplotype 5 (H5, tagged by SNP 13888T/G, rs2232354) of IL1RN (Odds ratio=3.9; 95% confidence interval: 1.6 to 9.7; P=0.002). No risk was associated with haplotype 3 of IL1RN, which contains the frequently examined allele 2 variant of the intron 2 VNTR. CONCLUSIONS: We found that IL1RN-H5H5 carriership increases the risk of venous thrombosis.

Plasma coagulation factor levels in venous thrombosis.

High plasma levels of several coagulation factors have been described to be associated with an increased risk of venous thrombosis. However, the mechanisms underlying these associations, as well as those involved in the regulation of plasma levels of coagulation factors, are mostly unknown. Whether these factors should be included in the workup of patients with venous thrombosis remains to be determined. In this review, we discuss the present knowledge on the effects of plasma levels of coagulation factors on the development of venous thrombosis. Furthermore, we review recent findings and ideas on the mechanisms through which elevated plasma coagulation factor levels may influence thrombosis. Finally, we enter into the matter of the possible determinants of elevated plasma levels of coagulation factors.

ABO blood group genotypes, plasma von Willebrand factor levels and loading of von Willebrand factor with A and B antigens.

ABO blood group is a genetic determinant of von Willebrand factor (VWF) levels. We investigated the effect of ABO genotypes on VWF and factor VIII (FVIII) levels and on the degree to which VWF is loaded with A- and B-antigens, expressed as normalized ratios, nA-ratio and nB-ratio, respectively, in the Leiden Thrombophilia Study, a large case-control study on venous thrombosis. We found that the ABO locus had an allele-specific, dosage dependent effect on VWF and FVIII levels and on the loading of VWF with A-antigen and B-antigen. The highest mean nA- and nB-ratios were found in A(1)A(1) and BB genotypes, respectively. Four A(1)O carriers had four 43-bp repeats in the minisatellite region of the ABO gene in stead of the expected one repeat. All had a reduced nA-ratio compared to A(1)O carriers with one repeat in their A(1) allele. The amount of A- and B-antigens expressed onVWF (nA-ratio and nB-ratio) explained about 18% (R(2)) of the variation in VWF levels.

Effect of oral contraceptives on thrombin generation measured via calibrated automated thrombography.

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 x 10(-3); 95% CI: -0.1 x 10(-3) to 2.0 x 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 x 10(-3); 95% CI: 6.3 x 10(-3) to 6.9 x 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.

Interleukin-6 induction of protein s is regulated through signal transducer and activator of transcription 3.

OBJECTIVE: The protein C anticoagulant pathway is an essential process for attenuating thrombin generation by the membrane-bound procoagulant complexes tenase and prothrombinase. In this pathway, protein S (PS) serves as a cofactor for activated protein C. PS circulates in plasma both in a free form and in complex with complement component 4b-binding protein (C4BP). C4BP is a known acute phase reactant, thereby suggesting a relation between PS and the acute phase response. Interleukin (IL)-6 has been shown to increase both PS and C4BP gene expression. Our objective was to study the regulation of PS gene expression by IL-6 in detail. METHODS AND RESULTS: IL-6 upregulates both PS mRNA and protein levels in liver-derived HepG2 cells. The promoter of the PS gene (PROS1) was cloned upstream from a luciferase reporter gene. After transfection in HepG2 cells, the luciferase activity was shown to be stimulated by the addition of IL-6. IL-6 exerts its effect through Signal Transducer and Activator of Transcription 3 (STAT3) that interacts with the PROS1 promoter at a binding site in between nucleotides 229 to 207 upstream from the translational start. CONCLUSIONS: IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.

Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease.

Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers. In contrast, single transfections of C2739Y or C2754W resulted in a quantitative VWF defect with low VWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both N- and C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain of VWF leads to a recessive, quantitative VWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.

Haplotypes encoding the factor VIII 1241 Glu variation, factor VIII levels and the risk of venous thrombosis.

Levels of factor VIII (FVIII) are associated with the risk of venous thrombosis. The FVIII variation D1241E has been reported to be associated with decreased levels of FVIII. Our objective was to study whether D1241E is associated with levels of FVIII and the risk of venous thrombosis and whether this association is caused by D1241E or another linked variation. We analyzed the association of three FVIII gene haplotypes encoding 1241E (further denoted as HT1, HT3, and HT5) with FVIII levels and thrombosis risk. This analysis was performed in the Leiden Thrombophilia Study (LETS). The control populations of two case-controls studies on arterial thrombosis in men and women, respectively, were used to confirm the effects observed on FVIII:C in the LETS. In men, HT1 was associated with a 6% reduction in FVIII:C and with a reduced risk of venous thrombosis [odds ratio 0.4 (CI95 0.2-0.8)]. Logistic regression showed that the risk reduction was only partially dependent of the reduction in FVIII levels. HT1 showed no effects in women on either FVIII:C or risk of thrombosis. The number of carriers of HT3 and HT5 was too low to make an accurate estimate of the risk of venous thrombosis. Neither HT3 nor HT5 showed effects on levels of FVIII:C. When we consider that all three haplotypes encoding 1241E show different effects on FVIII:C and thrombosis risk, it is possible that D1241E is not the functional variation. However, FVIII gene variations do contribute to both levels of FVIII and the risk of thrombosis.

Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden.

Two polymorphisms in the endothelial protein C receptor (EPCR) gene, 4600A/G and 4678G/C, have been reported to influence the risk of venous thromboembolism (VTE). The objective of this study was to assess whether these polymorphisms modify the risk of VTE in carriers of factor V (FV) Leiden. We genotyped 295 carriers of FV Leiden for these polymorphisms: 100 unrelated patients with a history of VTE (propositi) and 195 relatives (14 of them symptomatic) of 81 of the propositi. Spontaneous VTE events occurred in 71% of propositi carrying the 4678GG genotype, 65% carrying the GC, and 43% with the CC genotype. The mean age at the first onset was significantly higher in propositi carrying the 4678CC than in those with the GC or GG genotype (p = 0.046). Among the 276 carriers of FV Leiden from the 81 families studied, the 95 symptomatic members had similar 4600G allele and 4600AG genotype frequencies but significantly lower 4678C allele (p = 0.002) and 4678CC genotype (p = 0.004) frequencies than the 181 asymptomatic members. The probability of being free of thrombosis at age 40 was significantly higher in the 66 carriers of the 4678CC genotype (94%) than in the 138 carrying the GC (72%) or in the 72 with the 4678GG genotype (60%) (p < 0.001). Multivariate analysis showed that the 4678CC genotype reduced the risk of thrombosis in carriers of FV Leiden (OR = 0.31;95% CI = 0.16-0.83). The incidence of VTE was higher in the 195 relatives with FV Leiden than in the 133 without FV Leiden (OR = 4.7; CI = 1.3-7.2). These results show that carriers of FV Leiden with the 4678CC genotype have a significantly reduced risk of VTE compared with those carrying the 4678GG or GC genotype, probably due to the higherAPC levels previously observed in individuals carrying the 4678CC genotype.

C-reactive protein does not directly induce tissue factor in human monocytes.

OBJECTIVE: It is generally assumed that C-reactive protein (CRP) induces synthesis of tissue factor (TF) in monocytic cells, thereby potentially initiating intravascular blood coagulation. We aimed to elucidate the mechanism of CRP-induced TF expression in monocytes and monocyte-derived macrophages (MDMs) in vitro. METHODS AND RESULTS: Monocytes were isolated from the blood of healthy donors and cultured with or without CRP or lipopolysaccharide (LPS) to study the time course of TF antigen and TF mRNA expression. Addition of 100 microg/mL CRP did not result in a significant increase in TF antigen (range: 9 to 163 pg/10(6) cells, n=11) and TF mRNA (relative number of TF transcripts; N(TF)=0.01 to 0.33), when compared with nonstimulated cells (TF antigen 7 to 46 pg/10(6) cells, N(TF)=0.01 to 0.13). Variation of CRP concentration and exposure time did not affect the TF response. Similar results were obtained in monocytes cultured in suspension and in MDMs. In contrast, TF was strongly induced by 10 microg/mL LPS (TF antigen 1125 to 6120 pg/10(6) cells, N(TF)=5.94 to 23.43). Cultured monocytes did express FcRgammaII, a putative CRP receptor, and addition of CRP induced a 7-fold increase in the production of monocyte chemoattractant protein-1 (MCP-1). Interestingly, CRP addition to peripheral blood mononuclear cells (PBMCs) did result in TF expression on monocytic cells. CONCLUSIONS: The absence of TF induction after incubation of purified monocytes with CRP indicates that CRP is unable to induce TF expression in monocytes and MDMs directly. The presence of CRP-induced TF expression in PBMCs suggests that CRP can induce TF indirectly, probably through cross-talk between cells.

Candidate gene approach in association studies: would the factor V Leiden mutation have been found by this approach?

A re-emerging strategy in the search for disease susceptibility genes is the evaluation of candidate genes, which are thought to play a role in disease pathogenesis. Candidate genes are screened for single nucleotide polymorphisms (SNPs) in a case-control study. The factor V Leiden (FVL) mutation (1691G --> A in the F5 gene) is an important risk factor for venous thrombosis. We asked ourselves whether the FVL mutation would have been found using the candidate gene approach in the absence of prior knowledge of the haplotype structure of the F5 gene. We typed four SNPs in the F5 gene in the Leiden Thrombophilia study, that is, promoter (99930G --> A), exon 13 (55907A --> G), exon 16 (42855A --> G), and intron 19 (37833T --> G). These SNPs were known to have different population frequencies, making their presence in distinct haplotypes likely. None of these SNPs has previously been associated with venous thrombotic risk. Subsequently we derived haplotypes. One haplotype was clearly more frequent in patients than controls (GAAT; 20 versus 9%), suggesting that a polymorphism in or near the F5 gene in this haplotype is associated with an increased thrombotic risk. If we had sequenced the F5 gene in patients homozygous for this haplotype, in order to locate the possible causal polymorphism, we would have found that 16 (76%) patients were homozygous or heterozygous for a missense mutation in exon 10 (1691G --> A), which predicts the replacement of Arg506 by Gln in one of the cleavage sites for activated protein C, a mutation that we now know as the FVL mutation.