Autoactivation of human blood coagulation factor XII on dextran derivatives of different molecular weight

We prepared a derivative of dextran T40 (average Mr 43,000) from which fractions of different Mr but with equal charge density were obtained and tested for their ability to promote autoactivation of human blood coagulation factor XII. The mechanism of autoactivation appeared dependent upon the Mr of the polymer used. Thus, with polymers of 38,000 Mr or higher only -factor XIIa was formed and the reaction could be completely described in terms of a simple second-order mechanism of autoactivation. With smaller polymer molecules β-factor XIIa became a major reaction product and as a result of this the autoactivation kinetics did not adhere to the second-order mechanisms thus far described.     

A new assay for the determination of factor XII in plasma using a chromogenic substrate and a selective inhibitor of plasma kallikrein

A new chromogenic assay for factor XII in plasma was designed by the use of the soluble activator Kalli-plastin, the substrate H-D-HHT-Gly-Arg-pNA and a synthetic inhibitor of plasma kallikrein (Pefabloc PK). The assay was carried out in one and the same cuvette and did not require factor XII-deficient plasma. There was a good correlation between F XII coagulant and amidolytic assays.     

The effect of surfactants on the interaction of factor XII with surfaces

Factor XII (Hageman factor) binds irreversibly to plastic as well as glass surfaces. In dilute solution this leads to significant losses of protein onto the walls of tubes, etc. This could be prevented most efficiently by the presence of surfactants in solution. Rinsing the surfaces after contact with the protein, or precoating the surfaces were less effective. Triton X-100 was found to be non-denaturing and effective at concentrations as low as 0.001%. In its presence higher specific activities were obtained in spectrophotometric assay. The autoactivation of factor XII exposed to glass was inhibited but not prevented by Triton X-100. Similar effects on the binding and assay of (Pre) kallikrein were also found.     

Functional correlation between kallikrein and factor XII activated in human plasma

Plasma kallikrein and FXIIa were assayed in acetone-treated human citrated plasma (CPLa) with the chromogenic peptide Bz-Ile-Glu-Gly-Arg-pNA (S-2222) as substrate. In end point assays with short incubation periods (1–10 min.) nearly all kallikrein present could be blocked by a low concentration of soybean trypsin inhibitor (STI). In 30 min. assays the main part of the kallikrein was recovered in a functional state not inhibited by STI, and at the same time the level of FXIIa (as amidase activity blocked by corn inhibitor, C.I.) was reduced to about 2/3 of the initial value. The formation of an association between FXIIa and kallikrein is suggested. In fractions from gel filtration of CPLa kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassays, and HK and FXII were studied in PAGE immunoblot experiments. Kallikrein appeared as one peak together with HK (gel mol. wt. 300 KD), about 40% of HK was free (220 KD), and no FXII was observed in the kallikrein or HK peaks, but in two areas corresponding to 78–79 KD and 39–42 KD. When experiments, however, were carried out with plasma acetone-activated and gel filtered in the presence of benzamidine (5 mM), part of the amidase activity present in kallikrein peak fractions was blocked by C.I. This observation supports the above suggestion of an association between FXIIa and kallikrein.     

Preparation of β-XIIa (Hageman factor fragment) from human plasma

β-Factor XIIa (β-XIIa, $\text{M}$_r ～30,000) was isolated from human plasma by a procedure which utilized, as an initial step, the adsorption of Factor XII to celite. Activation of Factor XII and subsequent release of β-XIIa was brought about by the proteolytic action of co-adsorbed kallikrein. Two successive chromatographic procedures were then used to achieve a final purification of 4,420-fold over plasma and an overall yield of 2.3 mg of β-XIIa per liter.     

A simple and rapid method to study the association of the contact proteins of blood coagulation

Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The “mobility shift” assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.     

The mechanism by which the light chain of cleaved hmw-kininogen augments the activation of prekallikrein,factor XI and hageman factor

HMW-kininogen binds prekallikrein and factor XI to form two bimolecular complexes in plasma; upon addition of certain negatively charged surfaces, it functions as a coagulation cofactor and facilitates the activation of Hageman factor, prekallikrein and factor XI. We have investigated the mechanism by which HMW-kininogen functions in each of these steps. HMW-kininogen was found to augment the binding of prekallikrein and factor XI to kaolin in a plasma system sùggesting that the prekallikrein-HMW kininogen complex and the factor XI-HMW kininogen complex are attached to surfaces via HMW-kininogen. The attachment was shown to be mediated by the light chain derived from cleaved HMW-kininogen and is consistent with previous data demonstrating that the light chain is the coagulant part of the molecule. Prekallikrein can be readily activated when bound to the surface by this HMW-kininogen linkage; however, when an equal quantity of prekallikrein was bound directly to the surface, it was not activatable even after the addition of HMW-kininogen. Thus, binding of prekallikrein (and factor XI) to the surface via HMW-kininogen appears to place them in a conformationally favorable position for activation by activated Hageman factor. This appears to be the major function of HMW-kininogen as a coagulation cofactor. In addition, HMW-kininogen attachment of prekallikrein (kallikrein) to the surface is a reversible interaction which is not the case when direct binding to the surface occurs. In this fashion, a portion of the kallikrein formed can dissociate and interact with other Hageman factor molecules. These data suggest that the effect of HMW-kininogen upon Hageman factor activation is indirect and acts to increase the effective concentration of kallikrein available for Hageman factor cleavage.     

Diagnostic efficacy of the D-Dimer assay in disseminated intravascular coagulation (DIC)

The D-Dimer (D-D) assay for measuring cross-linked fibrin degradation products is now available for the clinical laboratory. We combined this assay with other tests to assess patients with diagnosed or suspected DIC. Also, a small group of patients (20) with deep venous thrombosis (DVT) were studied. The D-D test, antithrombin-III assay, FDP titer, fibrinopeptide-A level, protamine sulfate test, fibrinogen, prothrombin time, and activated partial thromboplastin time were used. The D-D test was abnormal in 93.7%, the AT-III level was abnormal in 87.5%, the fibrinopeptide-A level was abnormal in 89.5%, and the FDP titer was elevated in 83.7% of patients with DIC. When assessing patients found not to have confirmed DIC the D-D assay was abnormal in 20%, the AT-III level was abnormal in 6%, and the fibrinopeptide-A level was elevated in 13%. We conclude the D-Dimer assay to be a useful molecular marker of hemostasis in diagnosing DIC and this test will often discriminate between those patients with or without DIC, especially when used with the AT-III and fibrinopeptide-A assays. Of the battery of tests used in this study, the most useful, in descending order of efficacy, appear to be the D-dimer assay (93.7% abnormal), the fibrinopeptide-A titer (89.5% abnormal), the AT-III level (87.5% abnormal), and the FDP titer (83.7% abnormal).     

A study of hemostasis in ischemic cerebrovascular disease: IV. A five year follow-up of some blood coagulation parameters also including fibrinopeptide A,factor XII and prekallikrein

Twentyseven young adults (mean age 46) with ischemic cerebrovascular disease (ICD) were reinvestigated about 5 years after discharge and compared to 67 healthy controls. Factor VIII related antigen was again found significantly (p < 0.005 and p < 0.001) increased in male as well as female patients and a significant (r = 0.66, p < 0.001) correlation was found with earlier data. Factor VIII biological activity was again found increased, significantly (p < 0.001) in males. In contrast to earlier results antithrombin antigen and activity were significantly (p < 0.001) decreased in males. This finding and decreased levels of factor XII in female patients (p < 0.001) and of prekallikrein in male patients (p < 0.01) could reflect disturbed regulatory functions or possibly constitutional differences. As in most subjects no increase of fibrinopeptide A was found, there was no sign of continuous activation of the whole coagulation sequence. Since hemostatic abnormalities were unrelated to acute phase reacting proteins they were obviously of a different nature than unspecific response to tissue damage and acute stress. High levels of factor VIII and low levels of antithrombin imply that the coagulation system could be more easily activated when other factors coincide, e.g. intimal lesions in carotide arteries.     

A human factor VIIa concentrate and its effects in the hemophilic a dog

Properties of a new concentrate of FVII/FVIIa, obtained by adsorption onto an inorganic adsorbent followed by a chromatographic step onto Q-Sepharose using a by-product of routine fractionation as starting material,are described.This fraction contains only small amounts of the other components of the prothrombin complex but it is enriched in Proteins C and S.This preparation is essentially free of FVIIICag. It has been submitted to animal experiments including these carried out on normal and hemophilic A dogs.A normalization of the buccal bleeding time was seen after injection of a minimal dose of 4 uFVIIa/kg.No adverse reaction was observed at any dose employed.This concentrate might thus be indicated for the treatment of Haemophilia. A patients with inhibitors. Its viral inactivation has been achieved.     

A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS), were incubated with 50 μl of diluted normal human plasma (NHP) and 50 μl of Factor Xa-specific chromogenic substrate (CBS 31–39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31–39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10⁴ EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31–39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.     

Biosynthesis of factor V by normal adult rat hepatocytes

The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture. Two culture conditions were compared. A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium. Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays. Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins. Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells.

Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity. In contrast, treatment of cell extracts did increase the coagulant activity. This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form.

These data provide evidence for synthesis and secretion of an hepatocytic factor V.     

Assay for Hageman factor (factor XII) in human plasma by means of chromogenic substrate for plasma kallikrein

Determination of factor XII was performed in a test system where plasma samples were diluted (1/400-1/3200) in buffer and factor XII deficient plasma. Diluted Cephotest^R was used as activator and after 10 min activation time chromogenic substrate H-D-Pro-Phe-Arg-pNA was added. The assay was adopted to an enzyme analyzer. The reproducibility of the method was acceptable with a coefficient of variation of 7.8% in the normal range. The correlation of this method to a one-stage clotting assay for factor XII was good, r 0.79. The preliminary reference interval was calculated to be 52–147% of normal (mean+ 2 S.D.).