Platelets from a platelet factor 3-deficient patient, which was first described by Weiss et al (Am J Med 67:206, 1979), were found to be equally impaired in their ability to promote factor X and prothrombin activation. Compared to normal platelets, the patient's platelets showed upon stimulation with thrombin plus collagen a much slower generation and a considerably lower level of platelet prothrombin- and factor X-converting activities. Treatment of stimulated platelets with phospholipases revealed a decreased exposure of negatively charged phospholipid at the outer surface of the patient's platelets, relative to control's. We suggest that the combined impairment of prothrombin- and factor X-converting activities in this patient is due to a defect in the mechanism by which phosphatidylserine becomes exposed at the outer surface of stimulated platelets.

SummaryThe biological disappearance rate of coagulation factors II, VIT, IX and X from plasma is decreased in myxoedema and increased in thyreotoxicosis. In patients with elevated body temperatures, an increase of up to three times the normal rate was observed. The clinical implications of these findings are briefly discussed.

SummaryWe studied the mode of action of the low molecular weight heparin PK10169 and two of its constituent fractions: EMT 966 High Molecular Weight Fraction and EMT 967 Low Molecular Weight Fraction.EMT 966 like standard heparin, acts primarily on thrombin formed and not on prothrombinase (S type heparin). In contrast EMT 967 has no direct effect on thrombin. At high concentrations, it inhibits the prothrombinase complex (P type heparin). PK10169, that contains the two EMTs shows both activities: anti thrombin and antiprothrombinase (mixed type heparin).The addition of increasing amounts of EMT 967 to a constant amount of EMT 966 does not influence the breakdown constant of endogenous thrombin which is determined by the concentration of EMT 966 only. This demonstrates the absence of competition for AT III between the two components of PK10169.In platelet rich plasma, EMT 966 inhibits and postpones thrombin generation more efficiently than unfractionated heparin, probably because it is less sensitive to neutralization by platelet components (platelet factor 4). Amounts of EMT 967 that hardly inhibit thrombin generation in platelet rich plasma enhance the effect of EMT 966 probably by neutralizing platelet factor 4.

In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the alpha(2)macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment.

SummaryWhen normal citrated plasma is stored at 37° C and pH 7.8 the factor VIII activity drops to about 50% of its initial value during the first 8-12 h. In the following 4 days practically no further drop in activity is found. If the logarithm of the factor VIII activity is plotted against time a curve is obtained which can be described as biphasic. To explore the underlying mechanism of this phenomenon the influence of temperature, pH, Ca++ concentration and some other clotting factors was investigated. Between temperatures of 21° C and 45° C the inactivation of factor VIII was biphasic, the decrease of factor VIII being faster in both phases at higher temperatures. The inactivation at these temperatures showed a Q10 of about 2. At 52° C nearly all factor VIII activity disappeared within 8 h. Possibly the precipitation of fibrinogen at this temperature is of influence. Between pH 6.4 and 8.5 the decrease in factor VIII in the first phase was obviously slower at lower pH and the level of the second phase maintained at a higher factor VIII activity. No alteration of the normal inactivation pattern was seen in plasma from patients with congenital deficiencies of factors XII, IX or V or in normal plasma adsorbed with BaS04 which has factors II, VII, IX and X markedly decreased, nor was there any difference between platelet rich and platelet poor plasma. Low calcium concentrations (Resinplasma) markedly increased the rate of inactivation in the first phase, but did not influence the second phase.Four hypotheses are given to explain the biphasic inactivation of factor VIII: a) The presence of an inactivating substance in the first phase or a stabilizing factor in the second phase of factor VIII inactivation. b) The existence of two independent substances with factor VIII activity with different inactivation rates. c) Reversible denaturation of factor VIII in one or more steps. d) Factor VIII exists in plasma in two interdependent molecular forms. It is discussed that in view of the results of the experiments hypotheses a and b are not very likely. At present we cannot differentiate experimentally between c and d.