An improved assay for quantification of urinary porphyrins by use of second-derivative spectroscopy is described. A new method for calculation of the porphyrin concentration is developed and the whole procedure is computerized. Acidified urine samples can be assayed within a few minutes by using this method. Precision and recoveries for both uro- and coproporphyrin are good. The method is presented as a very fast and accurate assay for the screening and quantification of urinary porphyrins.

A mechanized factor X assay was tested in 2222 plasma samples of patients being treated with oral anticoagulants. The correlation coefficient between this assay and Thrombotest was 0.78. The therapeutic range for factor X amidolytic activity was 150 to 300 units/l. Amidolytic factor X activity and Thrombotest provide similar information about the state of anticoagulation within the same patient, including patients that are not well balanced. The ranges for factors II, VII, IX and X clotting activity in 57 patients on long-term therapy (Thrombotest within the therapeutic range between 190 and 95 s) were 80-300, 70-600, 40-420 and 50-330 units/l, respectively. The range for factor X amidolytic activity in this group of patients was 150-470 units/l.

We tested a mechanized amidolytic factor-X assay in 2222 patients on long term anticoagulant therapy. A good correlation was found between this assay and the routinely performed Thrombotest (r= 0.78). With a therapeutic range between 150 and 300 units/L of factor-X amidolytic activity we obtained the same information about the state of anticoagulation in 81 % of these 2222 patients. Factor-X activity was assayed in 32 stable anticoagulated patients together with Thrombotest on at least three subsequent occasions in each patient. A fairly constant Thrombotest-factor X ratio was observed.(mean 0.38±0.06) Changes in Thrombotest and factor-X amidolytic activity ran consistently parallel in a group of 20 unstable anticoagulated patients.(mean ratio 0.45±0.08) Changes in Thrombotest were occasionally more pronounced than changes in factor-X activity in some patients, which could be due to the high turnover of factor VII. Relative very high or low factor-VII levels as compared to the other vitamin-K dependent factors disturbed the balance between Thrombotest and factor X. The therapeutic consequence in these unbalanced states is, that the prescribed dosage scheme of the anti-vitamin K drug could depend on the very moment of blood sampling. As a result of the high turnover of factor VII, bloodsampling one day earlier or later would then result in another dosage scheme of the drug. Therefore it might be concluded that oral anticoagulant therapy, guided by a factor-X assay could result in more stable long term treatment. Large prospective comparative trials are required to support this hypothesis.

We describe the effect of pH and NaCl concentration on the activation of factor X in plasma by Russell's viper venom and on the amidolytic activity of the activated enzyme towards factor Xa-sensitive chromogenic substrates. An increasing NaCl concentration results in a decrease in the activation rate of factor X by Russell's viper venom, whereas in this step no pH effect is observed. Increasing NaCl concentrations decrease the Km and Vmax for both the factor Xa-sensitive chromogenic substrates, S 2222 and S 2337. Km values were lowest between pH 7.8 and 8.6; Vmax increased with increasing pH. Comparison of NaCl and KCl in the activation step as well as in the amidolytic step shows that the observed effect is specific for Na+, not just an effect of ionic strength. No difference was detected between activation by the crude venom or its purified factor X-activating enzyme. Also, values with the substrates S 2222 or S 2337 were the same in the mechanized amidolytic factor X assay.

We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

A new chromogenic method has been developed and rigorously standardized for the estimation of factor VII in defibrinated diluted plasma. This method employs a mixture of CaCl2-rabbit brain thromboplastin as activator, diluted factor VII deficient plasma as source of factor X and the chromogenic substrate S2222 for the measurement of factor Xa. The chromogenic method was insensitive to cold- and kaolin-induced activation of factor VII, this in contrast to the one-stage clotting assay. Results obtained with the chromogenic method revealed good correlation with the clotting method in 33 normal subjects, in 42 patients on oral anticoagulant therapy and in five patients with severe congenital factor VII deficiency. A good correlation was also obtained with 'Thrombotest'. Comparative estimation of factor VII and of factor VII cross-reacting material in supernatants of BaCl2 adsorbed plasma of coumarin treated patients revealed that the chromogenic method does not measure decarboxy factor VII. Detailed investigations revealed a half life for decarboxy factor VII of 2.1 +/- 0.6 h.

A group of 186 patients with Transient Ischaemic Attacks (TIA) or cerebral infarction (CI) was found to demonstrate in vitro Spontaneous Platelet Aggregation (SPA) in 39% of those studied. Of the 176 normal subjects studied the incidence on in vitro SPA was found to be 5%. Further investigation of the phenomenon of SPA revealed that: 1. it is associated with ADP-hyperaggregability, i. e. the threshold concentration to induce second wave aggregation is decreased; 2. it is dependant on the increase in pH which occurs in platelet-rich plasma stirring in an aggregometer while concurrent ADP-hyperaggregability is independant of this change in pH; 3. it is associated with malondialdehyde production and the release of endogenous 5-hydroxytryptamine; and that 4. in addition Km and Vmax values for [14c]-5HT incorporation are normal; and that 5. no gross abnormalities of the platelet membrane glycoproteins were apparent although occasionally glycoprotein III was found to be increased. This study demonstrates abnormal platelet behaviour in patients with TIA and CI where the enzyme system involved in thromboxane production is sufficiently stimulated, by stirring alone, to induce aggregation of platelets and the release reaction. Acetylsalicylic acid abolishes SPA and prolongs the bleeding time with similar characteristics as has been described for normal individuals. Plasma beta-thromboglobulin levels are significantly increased in the patients studied. However, no correlation was established with the incidence of in vitro SPA.