Factor V Leiden

In 1987-89, the ARIC Study recruited to a baseline examination a cohort of 15,792 men and women aged 45-64 years, predominantly whites or African Americans, from four U.S. communities (12 (link)). Participants were re-examined in 1990-92 (93% response), 1993-95 (86%) and 1996-98 (80%). Participants in the ARIC Visit 4 examination serve as the cohort for the present analysis.
CRP was measured in 2008 on plasma frozen at −70°C from Visit 4 by the immunoturbidimetric assay using the Siemens (Dade Behring) BNII analyzer (Dade Behring, Deerfield, Ill), performed according to the manufacturer's protocol. Approximately 4% of samples were split and measured as blinded replicates on different dates to assess repeatability. The reliability coefficient for blinded quality control replicates of CRP was 0.99 (421 blinded replicates). Body mass index was assessed as weight (kg) in a scrub suit divided by height (m) squared. Statins were assessed by reviewing participants' medication containers. After Visit 4, cholesterol-lowering medications were self-reported during annual telephone contact. Factor VIII_c and aPTT were not measured at Visit 4 so the Visit 1 value (3 (link), 13 (link)) was used. Factor V Leiden and the prothrombin G20210A polymorphism were not measured in the whole ARIC cohort.
Participants were followed from Visit 4 (1996-98, n = 11,573) through 2005 to identify hospitalized VTE events. These were validated by physician review using a standardized protocol (14 (link)). A total of 263 VTE events were identified, of which only 7 had been included in our previous analysis of baseline CRP and VTE through June 1997 (3 (link)). Excluding these 7 events had no impact on this analysis, so we chose to include them.
Our hypothesis was that CRP would be associated positively with VTE incidence. From the 11,573 participants at Visit 4, we excluded 320 who were missing CRP; 331 with CRP values >20 mg/L, due to possible acute phase response; 342 who had a prior history of VTE; or 204 who were taking warfarin. This left 10,505 at risk: 8,219 whites, 2,255 African Americans, and 31 others, who were grouped with African Americans for this analysis. Follow-up time ended when the participant had a VTE, died, was lost to follow-up, or else until December 31, 2005. Cox proportional hazards regression was used to model the association between CRP and VTE incidence, and to derive hazard ratios and 95% confidence intervals. Hazard ratios were calculated for each of the four highest quintile groups compared with the first, but also for high CRP categories (90^th or 95^th percentile) versus all others, to study the possible impact of high CRP on VTE. Covariates included previous VTE risk factors measured in the whole ARIC cohort, measured at Visit 4 unless otherwise specified: age (continuous), race (African American, white), sex/hormone replacement therapy (men, women taking HRT, women not taking HRT), diabetes (yes, no), body mass index (continuous), Visit 1 factor VIII_c, and Visit 1 aPTT. Other factors related to CRP (e.g., smoking, lipid levels, physical activity) were not VTE risk factors in ARIC, and thus not included.

All animal studies were done under Medical College of Wisconsin Institutional Animal Care and Use Committee-approved protocols. Wild-type C57Bl/6 mice were purchased from Harlan SD (Indianapolis, IN) and Factor IX-null mice were from Jackson Laboratories (Bar Harbour, ME). Factor VIII-null mice were kindly supplied by Dr. Haig Kazazian and Factor V Leiden mice were from Dr. David Ginsburg. Mice were anesthetized with intraperitonal pentobarbital (50 mg/kg) with maintenance dosing as required. Simple skin incisions permitted vessel exposures of the jugular vein (for intravenous injections), and of the carotid arteries, (with an external diameter of 0.3-0.5 mm) and femoral veins (diameter, 0.5-1.0 mm). Labeled platelets (up to 1 × 10⁷ injected per mouse) or fluorophore-labeled antibodies/proteins were injected into the jugular vein in volumes up to 100 μL, 3-5 minutes prior to thrombus induction; labeled anti-fibrin was used in the quantitative studies at amounts of 10-20 μg per mouse. Electrolytic injuries were induced on the surface of the carotid artery with a 30-second, 3-volt direct current application, touching the surface of the vessel with the blunt end of a 140-micron-diameter steel needle (Surgical Specialties Inc., Reading, PA) connected to the anode, completing the circuit by contacting local subdermal tissue with the cathode. A similar injury was made on the femoral vein, touching the surface for 30 seconds, 1.5 volts, with a 70-micron blunt-end needle. Applications under similar conditions with wires of copper, zinc, silver, or titanium did not yield detectable clots, supporting an iron ion-mediated electrolytic injury of clot induction, operating similarly as ferric chloride models of clot induction.^{3 ,4 (link)} A modified FeCl₃ model was also used for comparison: the corner of a piece of filter paper soaked in a 20% FeCl₃ solution was touched to the surface of the carotid artery for 30 seconds, comparable to the contact time and footprint used for the electrolytic injuries. Several other methods of thrombus induction were evaluated, which showed less consistency; these are described and presented in the Supplemental Section.