Quantitative immunoblotting assay of blood coagulation factor XII

The immunoblotting technique was applied to the study of Factor XII (F.XII) in plasma. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma followed by electroblotting of the electropherograms to nitrocellulose (NC) membranes and immunologic detection by a double antibody technique was used. ¹²⁵-F.XII was transferred to the NC membrane in amounts proportional to the amount applied to the gel provided that a constant amount of carrier protein was present. Based on this, a quantitative assay was developed using either normal plasma or F.XII dilutions in F.XII-deficient plasma as standards. The measurement of F.XII antigen by immunoblotting was reproducible and gave values similar to those obtained by radial immunodiffusion. Two normal plasma pools contained 26 and 29 μ/ml of F.XII according to the immunoblotting assay. Compared to other immunoassays, immunoblotting has the advantage of directly estimating the apparent molecular weight (MW) of the protein of interest. Thus, we could confirm the normal apparent MW (80,000) of a F.XII-like molecule previously isolated from a cross reacting material (CRM)-positive F.XII-deficient plasma. None of eight CRM-negative F.XII-deficient plasmas showed an 80,000 MW immunoreactive molecule. However, five of these eight plasmas had a faint autoradiographic band at 115,000 MW that was similarly seen in only three out of 43 individual normal plasmas. The nature of this 115,000 MW band remains to be defined.     

The plasma kallikrein–kinin system and risk of cardiovascular disease in men

BACKGROUND: The plasma kallikrein-kinin system (PKKS) has been implicated in cardiovascular disease, but activation of the PKKS has not been directly probed in individuals at risk of coronary heart disease (CHD) or stroke. OBJECTIVE: To determine the involvement of the PKKS, including factor XI, in cardiovascular disease occurring in a nested case-control study from the Second Northwick Park Heart Study (NPHS-II). METHODS AND RESULTS: After a median follow-up of 10.7 years, 287 cases of CHD and stroke had been recorded and 542 age-matched controls were selected. When FXIIa-C1 esterase inhibitor (C1-inhibitor) concentrations were divided into tertiles (lowest tertile as reference), the odds ratios (ORs) at 95% CIs for CHD were 0.52 (0.34-0.80) in the middle tertile and 0.73 (0.49-1.09) in the highest tertile (P = 0.01 for the overall difference; P = 0.01 for CHD and stroke combined). For kallikrein-C1-inhibitor complexes, the ORs for stroke were 0.29 (0.12-0.72) and 0.67 (0.30-1.52) in the middle and high tertiles, respectively (P = 0.02). FXIIa-C1-inhibitor and kallikrein-C1-inhibitor complexes were negatively related to smoking and fibrinogen (P < 0.005). FXIa-inhibitor complexes correlated strongly with FXIIa-inhibitor complexes. CONCLUSIONS: Lower levels of inhibitory complexes of the PKKS enzymes and particularly of FXIIa contribute to the risk of CHD and stroke in middle-aged men. This observation supports the involvement of the PKKS in atherothrombosis.     

A patient with an interstitial deletion of the short arm of chromosome 6

The clinical history and subsequent progress of a child with an interstitial deletion in the short arm of chromosome 6 is described. This abnormality coupled with a reduced Hageman factor (Factor XII) led to an earlier publication which suggested that this gene was localized to the breakpoint region involved. A review of similar phenotypes from the literature is presented.     

Plasma contact factors as therapeutic targets

Direct oral anticoagulants (DOACs) are small molecule inhibitors of the coagulation proteases thrombin and factor Xa that demonstrate comparable efficacy to warfarin for several common indications, while causing less serious bleeding. However, because their targets are required for the normal host-response to bleeding (hemostasis), DOACs are associated with therapy-induced bleeding that limits their use in certain patient populations and clinical situations. The plasma contact factors (factor XII, factor XI, and prekallikrein) initiate blood coagulation in the activated partial thromboplastin time assay. While serving limited roles in hemostasis, pre-clinical and epidemiologic data indicate that these proteins contribute to pathologic coagulation. It is anticipated that drugs targeting the contact factors will reduce risk of thrombosis with minimal impact on hemostasis. Here, we discuss the biochemistry of contact activation, the contributions of contact factors in thrombosis, and novel antithrombotic agents targeting contact factors that are undergoing pre-clinical and early clinical testing.