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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1333-1339

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1333-1339.)
© 1996 American Heart Association, Inc.

Articles

In Vivo Demonstration in Humans That Large Postprandial Triglyceride-Rich Lipoproteins Activate Coagulation Factor VII Through the Intrinsic Coagulation Pathway

Angela Silveira; Fredrik Karpe; Hans Johnsson; Kenneth A. Bauer; Anders Hamsten

the Atherosclerosis Research Unit, King Gustaf V Research Institute (A.S., F.K., A.H.), and the Thrombosis and Haemostasis Unit (H.J.), Department of Medicine, Karolinska Institute, Stockholm, Sweden; and the Department of Medicine, Beth Israel Hospital and Brockton–West Roxbury Department of Veterans Affairs Medical Center, Harvard Medical School, Boston, Mass (K.A.B.). Dr Hamsten is a Career Investigator of the Swedish Heart-Lung Foundation.

Correspondence to Angela Silveira, PhD, Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska Hospital, S-171-76, Stockholm, Sweden. E-mail silveira@instmed.ks.se.


*    Abstract
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 *Abstract
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 down arrowResults
 down arrowDiscussion
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In vitro studies in purified plasma systems have suggested that triglyceride-rich lipoproteins such as chylomicrons, very low density lipoproteins, and their remnants promote activation of factor VII through activated factor XII (XIIa) and the intrinsic coagulation pathway. We specifically examined the roles of factors XII, XI, and IX in activation of factor VII during alimentary lipemia in vivo in humans and addressed the issue of whether generation of activated factor VII (VIIa) is accompanied by increased thrombin production. For this purpose XIIa, factor IX activation peptide (IXP), VIIa, prothrombin fragment 1+2 (F1+2), and thrombin-antithrombin complex (TAT) were determined in plasma samples taken before and 3, 6, and 9 hours after intake of a mixed meal type of oral fat load in 24 healthy men. The VIIa response to fat intake was also determined in 7 patients with single coagulation–factor deficiency, of whom 2 were deficient in factor XII, 2 in factor XI, and 3 in factor IX. Postprandial activation of factors IX and VII occurred in the healthy individuals, whereas the plasma levels of XIIa did not change in response to the test meal. Of note, plasma concentrations of F1+2 were unaltered during alimentary lipemia, and TAT levels showed a small decrease (P<.05) in the 3-hour sample compared with the fasting level, indicating that thrombin generation is not stimulated in the postprandial state, despite the generation of activated factor IX (IXa) and VIIa. Factor VIIa increased in the postprandial period in the 2 factor XII–deficient patients who underwent the oral fat tolerance test but appeared to remain unchanged in the factor XI– and factor IX–deficient patients. Therefore, the current concept that activation of factor XII plays a pivotal role in initiating the sequence of events linking postprandial lipemia to activation of factor VII is contradicted by the present study. Whether activation of factor XI by triglyceride-rich lipoproteins initiates these reactions needs to be demonstrated in future studies.


Key Words: factor VII • factor XII • factor XI • factor IX • alimentary lipemia


*    Introduction
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 *Introduction
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 down arrowResults
 down arrowDiscussion
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Factor VII is the first enzyme in the extrinsic pathway of blood coagulation. It is present in normal plasma at a concentration of about 450 µg/L,1 the major proportion of which circulates in the zymogen single-chain form. However, low levels of the activated double-chain form (around 1% of the factor VII mass) are also present and appear to serve a priming function for triggering of the clotting cascade.2 3 4 5 6 VIIa bound to TF initiates blood coagulation by activating factors IX and X,7 8 9 a process already observed in the 1960s by Josso and Prou-Wartelle10 and Nossel.11 In the presence of calcium and phospholipids, Xa activates factor VII, thus amplifying the coagulation response elicited on expression of TF.12 In parallel, XIIa and IXa generate VIIa by interactions with the zymogen through processes that do not require TF (the intrinsic coagulation pathway).13 14 15 The relationships between the activation of factor XII and generation of VIIa, first described by Soulier and Prou-Wartelle16 and then by Altman and Hemker,17 have now been carefully elucidated. In vitro factor IXa, generated through activation of either factor XI or prekallikrein by XIIa, is responsible for the major part of the TF-independent factor VII activation, and the rest seems to be controlled via direct activation by XIIa.18

Dietary studies in humans and animal models have established a connection between plasma concentrations of triglyceride-rich lipoproteins and factor VII coagulant activity, as measured by one-stage bioassays. For instance, addition of fat to the diet causes a rapid increase in factor VII coagulant activity, which has been interpreted to reflect an increased conversion of the zymogen to VIIa.19 20 21 22 23 24 In vitro studies in purified plasma systems have also suggested that lipoprotein particles such as chylomicrons, VLDL, and their remnants, carrying the appropriate FFA at a sufficient density of negative charge, promote contact activation of the intrinsic coagulation pathway and thereby of factors VII through XIIa.18 25 26 The generation and subsequent transfer of FFA from the triglyceride core to the phospholipid surfaces of the large triglyceride-rich lipoproteins through the action of LPL appear to play an important role in this sequence of events.27 28 The putative contact surface on the remnants of triglyceride-rich lipoproteins is probably provided by stearic acid.29 However, it is notable that the current concept of a central role of factor XII in the interactions between triglyceride-rich lipoproteins and the intrinsic coagulation pathway that lead to factor VII activation is based solely on in vitro studies in purified plasma systems.

We have recently demonstrated that factor VII is activated during alimentary lipemia in healthy human subjects and in patients with manifest coronary heart disease.30 31 The postprandial triglyceridemia was found to precede the activation of coagulation factor VII, and the increases in plasma triglycerides and the larger species of triglyceride-rich lipoproteins correlated quantitatively with the degree of factor VII activation. Furthermore, the rise in newly lipolyzed FFA from chylomicrons and chylomicron remnants was associated with the degree of factor VII activation. Alimentary lipemia was therefore considered an appropriate model to determine the mechanisms underlying activation of factor VII by triglyceride-rich lipoproteins in vivo in humans. In the present study we specifically examined the roles of factors XII, XI, and IX and addressed the issue of whether VIIa generation is accompanied by increased thrombin production.


*    Methods
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 *Methods
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Subjects
A total of 24 healthy men aged 35 to 45 years (41.8±1.9 years, mean±SD), along with 7 patients with single coagulation–factor deficiencies, were enrolled in this study. The healthy subjects were recruited from a population survey including 129 subjects of Northern European descent. Only individuals who were homozygous for the E3 allele32 and who had fasting plasma VLDL triglyceride and LDL cholesterol concentrations below the 90th percentiles of all participants in the survey (<1.95 and <4.70 mmol/L, respectively) were considered for the study. Recruitment procedures and representativeness have been described in detail previously.33 Four of the 24 men were smokers. Body mass index was below 28.0 kg/m2 in all subjects (24.1±2.1 kg/m2, mean±SD).

The patient group comprised seven subjects, of whom two were deficient in factor XII, two were deficient in factor XI, and three were deficient in factor IX (Table 1Down). The factor IX–deficient subjects had a severe bleeding diathesis and were on regular supplementation with human purified factor IX. The postprandial study was performed 3 days after the last injection.


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  		Table 1. Basal Levels of Factors XII, XI, X, IX, and VII in Single Coagulation Factor Deficiencies (n=7)

Oral Fat Tolerance Test
Participants were admitted early in the morning to the Clinical Research Unit for a mixed-meal type of oral fat tolerance test.34 They had been fasting for 12 hours and were asked to refrain from smoking during the fasting period. The test meal was well tolerated by all subjects. Participants were allowed to be ambulatory and drink water throughout the test period. Food and smoking were prohibited.

Blood Sampling
A fasting blood sample was taken before the test meal. Subsequent blood samples were drawn hourly for lipoprotein determinations and at 0, 3, 6, and 9 hours for coagulation analysis. For lipoprotein determinations, venous blood was obtained through an indwelling catheter in one arm and drawn into precooled sterile 10-mL tubes containing 0.12 mL of 0.34 mol/L tripotassium EDTA (Vacutainer, Becton Dickinson). The tubes were immediately put into an ice-water bath. Plasma was prepared within 30 minutes by low-speed centrifugation (1750g, 20 minutes, 1°C) and kept at this temperature throughout the preparation procedures. Sodium azide and the protease inhibitors PMSF (10 mmol/L, dissolved in isopropanol; Sigma Chemical Co) and aprotinin (1400 µg/mL, Trasylol, Bayer) were immediately added to the isolated plasma before fractionation of triglyceride-rich lipoproteins to final concentrations of 1.0 mmol/L, 10 µmol/L, and 28 µg/mL, respectively. For coagulation analysis, venous blood was obtained by antecubital venipuncture in the other arm with a 1.4-mm Wasserman needle (size DIM 1.4x45 mm, TSK Laboratories) and drawn into 10-mL plastic tubes containing 1 mL of 0.129 mol/L trisodium citrate. Plasma was recovered after centrifugation (2200g, 20 minutes, room temperature) and immediately stored at -80°C in small aliquots. Blood samples were collected without stasis or when necessary, with minimal stasis on the forearm after 10 minutes' supine rest. Blood for factor VII genotyping was drawn into the same type of sterile tubes as for lipid and lipoprotein analyses and immediately frozen at -80°C.

Coagulation Assays
Levels of factor XII, XI, X, IX, and VII were determined in the basal plasma samples of all patients with single coagulation–factor deficiencies. Factors XII, XI, and IX were determined with a clotting method using the silica activator PTT automate (Diagnostica Stago) and specific factor-deficient plasmas (Helena Laboratories) using an MLA Electra 900C automatic coagulation timer. Factor X was determined as factor Xa amidolytic activity with the use of the synthetic substrate benzoylisoleucyl-glutamyl-glycyl-arginine-para-nitroanilide, S-2222 (Chromogenix), after complete activation of factor X zymogen in the presence of Russell's viper venom and calcium. Factor VII concentration was determined with an enzyme immunoassay kit (Novoclone Factor VII EIA kit, a gift from Novo Nordisk A/S, Bagsvaerd, Denmark). Interassay coefficients of variation for the high and low reference plasmas were between 4% and 9% for all these analyses.

Levels of VIIa, XIIa, IXP, F1+2, and TAT were determined in plasma samples drawn before ingestion of the test meal and at 3, 6, and 9 hours thereafter in all healthy individuals and patients with single coagulation–factor deficiencies.

Plasma levels of VIIa were determined with a clotting assay using soluble recombinant truncated tissue factor (s-TF, lot CCIC III-94, a kind gift from Dr Peter Wildgoose, Novo Nordisk A/S),5 6 as described.30 Coagulation times were converted to VIIa concentration (µg/L) by comparison with a standard curve constructed from varying concentrations of purified recombinant factor VIIa (a kind gift from Prof Ulla Hedner, Novo Nordisk A/S). Data were collected on a Compaq Presario 425 microcomputer (Compaq Computer Corporation) and analyzed using the Windows Research Software supplied with the ACL-300 coagulometer (Instrumentation Laboratories), essentially as described.6 Intra-assay and interassay coefficients of variation were 3.9% and 9.1%, respectively. Control experiments were performed to exclude nonspecific effect of lipids in the postprandial samples on the VIIa assay. Addition of VLDL (purified from normal individuals) to the plasma levels attained in the postprandial state did not increase the rate of hydrolysis of the synthetic substrate N-methylsulphonyl-D-phenylalanyl-glycyl-arginine-para-nitroanilide acetate (Chromozym-tPA) by a preparation containing recombinant factor VII and recombinant VIIa at concentrations corresponding to those measured in plasma as well as rabbit brain cephalin, soluble recombinant truncated TF, and calcium at concentrations used in the clotting assay for determination of VIIa. The absence of any increase in VIIa in the factor XI– and factor IX–deficient patients studied, in whom a similar increase in postprandial triglyceride-rich lipoproteins was observed compared with patients without these coagulation factor deficiencies, is further evidence against nonspecific effect of lipids in the VIIa assay (see "Results").

XIIa was determined with a direct enzyme immunoassay that detects both {alpha}-XIIa and ß-XIIa in human plasma, available from Shield Diagnostics Limited. Intra-assay and interassay coefficients of variation were 3.3% and 8.1%, respectively, for a reference plasma in which the XIIa concentration was 1.98±0.20 µg/L. No XIIa antigen was detected in the plasma of the factor XII–deficient patients studied. IXP was measured by a double antibody radioimmunoassay.8 F1+2 and TAT were determined with sandwich-type enzyme immunoassays using commercially available kits (Enzygnost TAT Micro and Enzygnost F1+2 Micro, Behringwerke AG).

DNA Procedures
Genotyping was performed for the Arg-Gln polymorphism at amino acid 353 in factor VII, which is known to reduce the factor VII levels in plasma.35 36 Nucleated cells from frozen whole blood were prepared according to Sambrook et al,37 and DNA was extracted by a salting-out method.38 Enzymatic amplification was performed by PCR with 50 ng to 1 µg genomic DNA and thermostable Taq polymerase (Boehringer Mannheim Scandinavia) according to the manufacturer's instructions. Oligonucleotide primers for PCR were made by Pharmacia. The PCR reactions were performed in a GeneAmp PCR System 9600 (Perkin-Elmer). Oligonucleotide primers, cycle times, temperatures, and conditions for Msp I digestion and electrophoresis have been described in detail.35 The common M1 allele, coding for Arg353, gave bands of 205 bp and 67 bp, and the M2 allele, coding for Gln353, gave a band of 272 bp as described previously.35

Lipoprotein Fractionation and Determination of Apolipoproteins B-48 and B-100
Triglyceride-rich lipoproteins (chylomicrons, VLDL, and their respective remnants) were fractionated from plasmas drawn before ingestion of the test meal and at 3, 6, and 9 hours thereafter by cumulative rate ultracentrifugation.34

Samples containing isolated lipoprotein fractions were first delipidated in a methanol/diethylether solvent system and denatured, after which the apo B-48 and apo B-100 contents were determined by analytical SDS-polyacrylamide gel electrophoresis.39

The major plasma lipoproteins VLDL, LDL, and HDL were also determined on the fasting plasma sample by a combination of preparative ultracentrifugation and precipitation of apo B–containing lipoproteins followed by lipid analysis.40

Lipid Analyses
Total cholesterol41 and triglycerides42 were determined in plasma and in the major plasma lipoproteins obtained from the fasting blood sample on an Ultrolab (Pharmacia LKB Biotechnology AB) after extraction with chloroform/methanol.43 Plasma triglycerides were also measured on the fasting and postprandial samples by an enzymatic method (877557, Boehringer Mannheim Diagnostica).

Statistical Analysis
Conventional methods were used for calculation of mean, SD, and SEM. Coefficients of skewness and kurtosis were calculated to test deviations from a normal distribution. Logarithmic transformation was performed on the individual values of skewed variables, and a normal distribution of values was confirmed before statistical computations and significance testing. Spearman's rank correlation coefficients were calculated between XIIa, IXP, and VIIa and lipid and lipoprotein variables. Within-group comparisons of measurements made at various time points during the oral fat tolerance test were done by Student's paired t test. To estimate the overall responses of different variables present in the plasma during the entire 9-hour postprandial period, AUC for plasma measurements plotted against time after intake of the test meal were calculated.

Ethical Considerations
The experimental protocol was approved by the ethics committee of the Karolinska Hospital, Stockholm, Sweden. All subjects gave informed consent to participate in the study.


*    Results
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 *Results
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Basal Plasma Levels of XIIa, IXP, VIIa, Lipids, and Lipoproteins in Healthy Subjects
Table 2Down shows the plasma levels of XIIa, IXP, and VIIa and lipids and lipoproteins in the basal fasting plasma sample of the healthy subjects. Results for VIIa are presented separately for individuals with Arg-Arg and Arg-Gln genotypes, as factor VII activity is known to be influenced by this polymorphism.30 35 36 Correlation coefficients were calculated between coagulation factors and lipoprotein measurements in men who were homozygous for the factor VII Arg allele, whereas the limited number (n=4) of individuals with Arg-Gln genotype precluded a separate analysis in this group. For XIIa, a strong positive correlation was found with the HDL cholesterol level (r=.71, P<.01). In contrast, no fractions of triglyceride-rich lipoproteins related significantly to the XIIa concentration. Factor VIIa also correlated significantly with the HDL cholesterol concentration (r=.53, P<.05), whereas no significant correlations were found between IXP and the lipoprotein measurements made in the fasting state.


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  		Table 2. Fasting Plasma Levels of XIIa, IXP, VIIa, Lipids, and Lipoproteins in Healthy Subjects (n=24)

Responses of Plasma Triglycerides and Triglyceride-Rich Lipoproteins to the Oral Fat Load in Healthy Subjects
Whole plasma triglycerides increased significantly in response to the test meal, with peak levels attained after 4 hours (Fig l)Down. The concentrations of apo B-48 and apo B-100 in subfractions of triglyceride-rich lipoproteins showed similar patterns, with the exception of Sf 12 to 20 apo B-100, which decreased in the 3-hour sample (Fig 2Down). As previously discussed in detail,33 the response of plasma triglycerides and subfractions of triglyceride-rich lipoproteins to fat intake showed considerable heterogeneity, despite the fact that healthy normolipidemic subjects with an apo E3/E3 genotype were studied. The responses of plasma triglycerides to the oral fat load in the patients with single coagulation–factor deficiency did not differ from the responses that were observed in the healthy subjects (data not shown).



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  		Figure 1. Plasma triglyceride concentrations after ingestion of the oral fat load. Values are mean±SEM, n=24.




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  		Figure 2. Plasma apo B-48 (left) and apo B-100 (right) concentrations in subfractions of Sf>12 lipoproteins after ingestion of the oral fat load in healthy subjects. Apo B concentrations were determined separately in the Sf >400 ({bullet}), Sf 60 to 400 ({blacksquare}), Sf 20 to 60 ({blacktriangleup}), and Sf 12 to 20 ({circ}) lipoprotein fractions. Values are mean±SEM, n=24.

XIIa, IXP, and VIIa Concentrations and Measurements of Thrombin Generation During Alimentary Lipemia in Healthy Subjects
Plasma levels of XIIa did not increase in the healthy individuals in response to the test meal (Fig 3Down). A small but significant decrease in XIIa was, on the contrary, observed in the 6-hour sample compared with the baseline level (P<.05). Postprandial plasma levels of IXP peaked in the 3-hour sample, remained significantly increased in the 6-hour sample, and showed a significant decrease at 9 hours after fat intake compared with baseline (Fig 3Down). VIIa responses to the oral fat load are shown separately for healthy individuals with Arg-Arg and Arg-Gln genotypes (Fig 3Down). Activation of factor VII occurred in both groups (P<.01 and P<.05 for the {Delta}0 to 6-hour increase in VIIa in Arg-Arg and Arg-Gln individuals, respectively), maintaining in the postprandial state the differences in VIIa levels that existed in the baseline sample. Plasma concentrations of F1+2 did not change during alimentary lipemia (Fig 4Down). In contrast, TAT levels showed a small but significant (P<.05) decrease in the 3-hour sample compared with the fasting level (Fig 4Down). These data indicate that no increase in thrombin generation occurred during alimentary lipemia, despite the generation of IXa and VIIa.





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  		Figure 3. Responses of the plasma concentrations of XIIa (left, n=20), IXP (middle, n=22), and VIIa (right) to the oral fat load in healthy subjects. VIIa levels are shown separately for individuals with Arg-Arg ({circ}, n=20) and Arg-Gln ({bullet}, n=4) genotypes. Values are mean±SEM. *P<.05; **P<.01 compared with the baseline level.




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  		Figure 4. Responses of the plasma concentrations of F1+2 (left) and TAT (right) to the oral fat load in healthy subjects. Values are mean±SEM, n=24. *P<.05 compared with the baseline level.

Relations of IXP and VIIa to Plasma Triglycerides and Triglyceride-Rich Lipoproteins During Alimentary Lipemia in Healthy Subjects
Relations between postprandial levels of IXP and VIIa and plasma triglycerides and triglyceride-rich lipoproteins were determined among healthy subjects with the factor VII Arg-Arg genotype (Table 3Down). For IXP, significant positive correlations were found between plasma triglycerides and Sf>400 apo B-48 both for the 0- to 3-hour increases in plasma concentrations of these variables and for the total AUC. The 0 to 3-hour increase in plasma VIIa level also appeared to be related to the corresponding increase in plasma triglycerides and Sf>400 apo B-48. Similarly, significant correlations were also found between the 0 to 6-hour increases in VIIa and plasma triglycerides and Sf 60 to 400 apo B-48 (r=.47, P<.05 and r=.54, P<.05, respectively).


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  		Table 3. Correlation Coefficients Between Plasma Triglycerides and Large Triglyceride-Rich Lipoproteins and IXP and VIIa During Alimentary Lipemia in Healthy Subjects With Arg-Arg Factor VII Genotype

VIIa During Alimentary Lipemia in Coagulation Factor–Deficient Patients
VIIa increased in the postprandial period in both factor XII–deficient patients who underwent the oral fat tolerance test (Fig 5Down). In one patient, VIIa levels peaked in the 6-hour sample and returned to the baseline level in the 9-hour sample. In the other, VIIa peaked in the 9-hour sample, after a discrete gradual increase from the baseline level. Of note, the triglyceride response preceding the factor VII activation in this patient was delayed as well. In contrast, factor XI– and factor IX–deficient patients did not appear to show significant alterations in the plasma VIIa concentration during alimentary lipemia (Fig 5Down). The two factor XI–deficient patients had basal VIIa concentrations in the upper normal range or distinctly elevated, respectively, and comparable VIIa concentrations were not attained in the postprandial state by most of the healthy volunteers.





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  		Figure 5. Responses of the plasma concentrations of VIIa to the oral fat load in patients who were deficient in factor XII (left), factor XI (middle), or factor IX (right). Each line represents one patient.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We here report the finding of a joint activation of coagulation factors IX and VII in the postprandial state in humans, which was not accompanied by activation of factor XII. In addition, we observed that activation of factor VII was normal in factor XII–deficient patients who performed the oral fat tolerance test, whereas it was not discernible in both factor XI– and factor IX–deficient patients. Furthermore, the basal generation of thrombin was unaffected by increased plasma levels of VIIa.

Studies in vitro in purified systems have attempted to determine the molecular mechanisms underlying the activation of factor VII by triglyceride-rich lipoproteins.18 25 27 Chylomicrons, chylomicron remnants, and large VLDL particles, which accumulate in plasma in the postprandial state, have been proposed to be the responsible lipoprotein species, as they carry the appropriate FFAs at a density of negative charge sufficient for contact activation of factor XII.27 Subsequently, XIIa would activate factor XI to XIa, and factor XIa would in turn activate factor IX to IXa.18 Finally, factor IXa, and also factor XIIa, would activate factor VII to VIIa.18 However, two independent sets of experiments performed in vivo in humans in the present study have failed to confirm the participation of factor XII in the sequence of events leading to activation of factor VII in the postprandial state. First, XIIa was not generated in plasma in the postprandial state in healthy volunteers. Second, activation of factor VII was normal in factor XII–deficient patients. Instead, factors XI and IX appeared to be critical, because factor XI– and factor IX–deficient patients did not seem to activate factor VII in response to fat intake. Surprisingly high basal VIIa concentrations were encountered in the two factor XI–deficient patients. In fact, comparable VIIa levels were not attained in the postprandial period by most of the healthy volunteers. Further studies in larger groups of patients deficient in factor XI are needed to confirm the high basal VIIa concentration and the absence of postprandial activation of factor VII and to disentangle the underlying molecular mechanisms. The basal VIIa concentrations in the factor IX–deficient patients were also not as low as previously observed,5 44 probably because the levels of factor IX were not as severely reduced in our patients as in the patients in previous studies.

The increased concentrations of both IXP and VIIa in the postprandial period correlated with the plasma levels of large chylomicron remnants as well as with plasma triglycerides. This observation emphasizes the significance of the large intestinal lipoproteins and the role of dietary lipids in determining the plasma concentration of VIIa. The specific and sensitive coagulation assays combined with detailed analyses of chylomicron remnants and VLDL in the present study confirm and extend the recent work that has established a firm connection between diet, triglyceride-rich lipoproteins, and factor VII activity.19 20 21 22 23 24 Furthermore, the strong positive correlation between the HDL cholesterol concentration, which would be expected to reflect LPL activity,45 and the basal VIIa level supports the notion that LPL plays a crucial role in the activation of factor VII through the intrinsic pathway.27 28 Striking positive relationships have previously been demonstrated between nonfasting plasma concentrations of triglyceride-rich lipoproteins (chylomicrons, VLDL cholesterol, and VLDL triglycerides) and XIIa.46 These results contrast with the results of the present study, in which significant correlations were found between neither fasting nor postprandial triglyceride-rich lipoproteins and XIIa. In contrast, basal XIIa and fasting HDL cholesterol concentrations were positively related. This discrepancy could be partly accounted for by differences in study design and methods.

Despite a significant increase in the plasma concentration of VIIa, no thrombin generation occurred during alimentary lipemia, as we did not observe any increases in F1+2 and TAT in the postprandial plasma samples. This finding is in apparent contrast to a recent animal study in which infusion of relatively high concentrations of recombinant VIIa into normal chimpanzees resulted in significant increases in the plasma levels of Fl+2 and other markers of coagulation activation.9 This report also suggests that VIIa is prothrombotic through interaction with some endogenous TF sites, from where it competitively displaces factor VII.9 The discrepancy between our human data obtained during alimentary lipemia and the results of infusing recombinant VIIa into chimpanzees might depend on differences in the amounts of VIIa available for competition for saturated TF sites. Alternatively, the plasma concentrations of tissue factor pathway inhibitor (the Xa–dependent inhibitor of the VIIa-TF complex) may be sufficient to inhibit the VIIa-TF complexes generated postprandially in humans. Furthermore, it cannot be excluded that a slight increase in the basal generation of thrombin in the healthy human volunteers was left undetected by the F1+2 and TAT assays. However, our data are in agreement with studies of hemophilia B patients, in whom infusion of a highly purified factor IX concentrate resulted in an increase in VIIa concentration that was not accompanied by a significant rise in F1+2.44

The current concept, based on in vitro studies in purified plasma systems and previously not properly explored in vivo in humans, that activation of factor XII plays a pivotal role in initiating the sequence of events linking postprandial lipemia to activation of factor VII clearly is contradicted by the present study. Whereas factor XII activation appears not to be a prerequisite for generating VIIa, availability of factors XI and IX seemed to be required, and IXP levels in plasma were found to increase in response to fat intake. Whether activation of factor XI by triglyceride-rich lipoproteins initiates the reactions that produce VIIa during alimentary lipemia needs to be demonstrated in future studies.

Since no signs of increased thrombin generation were observed during alimentary lipemia, it could be argued that the intrinsic activation of factor VII would not be related to an increased risk of thrombosis. However, VIIa serves a priming function for triggering of the extrinsic pathway of blood coagulation. Increased generation of VIIa in the postprandial state through the intrinsic coagulation pathway therefore strengthens the potential for thrombin production in the event of plaque rupture with exposure of TF. The strength of the procoagulant force could thus be set in part by the activity state of the factor VII molecules, which is influenced by postprandial triglyceride-rich lipoproteins and by the number of exposed TF sites. Since most of our lives are spent in the postprandial state, alimentary lipemia may be seen as a clinically important procoagulant state that is likely to promote the formation of occlusive thrombi on fissured atherosclerotic plaques.


*    Selected Abbreviations and Acronyms
 
AUC = areas under the curve
F1+2 = prothrombin fragment 1+2
FFA = free fatty acid
VIIa, IXa, Xa, and XIIa = activated factors VII, IX, X, and XII
IXP = factor IX activation peptide
LPL = lipoprotein lipase
PCR = polymerase chain reaction
Sf = Svedberg flotation rate
TAT = thrombin-antithrombin complex
TF = tissue factor


*    Acknowledgments
 
This study was supported in part by grants from the Swedish Medical Research Council (8691), the Swedish Heart-Lung Foundation, the Marianne and Marcus Wallenberg Foundation, the Tore Nilsson Foundation, the Professor Nanna Svartz Fund, the Foundation for Old Servants, and National Institutes of Health grant PO1HC-33014. The soluble recombinant truncated tissue factor (given by Dr Peter Wildgoose), the purified recombinant VIIa (given by Prof Ulla Hedner), and the enzyme immunoassay kit were provided by Novo Nordisk A/S, Bagsvaerd, Denmark.

Received December 12, 1995; revision received April 4, 1996;
*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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