Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg 506 3 Gln (factor V R506Q

In the present paper we compare prothrombin-converting activities of platelets non-activated, or activated by collagen, thrombin or collagen plus thrombin in the absence and presence of added factor V,. In all experiments described, the rate of thrombin formation for platelets activated by the combined action of collagen and thrombin is greater than that of platelets stimulated by collagen or thrombin alone. The presence of added factor V, enhanced the rate of thrombin formation in all cases, but the higher activity observed with platelets stimulated by collagen plus thrombin remains. When platelets are activated by collagen plus thrombin in the presence of factor X, and prothrombin, a lag period of approximately 10 min is observed before the rate of thrombin formation reaches a steady state. Addition of an excess of factor V, in this experiment reduces the lag time to 3 min. This lag period is interpreted as the time required to generate extra binding sites for the prothrombinase complex at the platelet surface. These extra sites explain the difference in thrombin formation rate between these platelets and platelets activated by either collagen or thrombin only.The exposure of phospholipids at the platelet outer surface was studied with phospholipases after various activations of the platelets. It is demonstrated that activation by collagen plus thrombin is accompanied by increased susceptibility of platelet phospholipids towards phospholipase Az. Among these degradable phospholipids are 25 % of the phosphatidylserine and 30 % of the phosphatidylethanolamine. On the other hand, little or no phosphatidylserine is exposed at the membrane exterior of thrombin-treated or control platelets. We propose that the exposure of phosphatidylserine at the outer surface of platelets activated with thrombin plus collagen is essential for the rate enhancement of thrombin formation observed under these conditions. The possibility of a transbilayer movement of phospholipids in the platelet membrane as a result of the activation process will be discussed.One of the major functions of platelets in the process of hemostasis is to provide a catalytic surface for the formation of the intrinsic factor-X-activating complex and the prothrombinase complex (for a review, see [I]). Nesheim et al. [2] have shown that there is no appreciable difference in catalytic efficiency of the prothrombinase complex when phospholipid vesicles plus factor Va are substituted for activated platelets. Rosing et al. [3] showed that the role of phospholipids in the prothrombinase complex is to decrease the K,,, for prothrombin below its plasma concentration, whereas factor V, strongly increases the V,,, of the reaction [2,3]. Recently, similar results were found for the role of phospholipids in the intrinsic factor-X-activating complex [4].It has been demonstrated that the cytoplasmic surfaces of both erythrocytes and platelets, as well as liposomes prepared from phospholipids present in the inner leaflet of the plasma membrane of these cells, possess a str...

SummaryA method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording of p-nitroaniline (pNA) production in a 2:3 diluted plasma. A chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ68), is used that is specifically split by thrombin but at a low rate. The thrombin that appears and disappears in the plasma does not split more than 5% of the substrate added, so the rate of substrate conversion is in good approximation proportional to the amidolytic activity in the plasma over the entire period of thrombin generation. The course of the enzyme concentration can be calculated from the amidolytic activity curve. It is shown that the thrombin generation curves obtained in this way are essentially identical to those obtained via the classical subsampling method.The presence of SQ 68 influences the amount of free thrombin that appears in plasma because it competitively inhibits the inactivation of thrombin by AT III and α2 macroglobulin. The inhibition of the thrombin peak by heparin, relative to an uninhibited control, remains unaltered by the presence of the substrate.From the course of thrombin activity and the prevailing decay constants, the course of prothrombin conversion velocity can be calculated. Prothrombin conversion was seen to be inhibited at high (>500 μM) substrate concentrations only, and experimental conditions are found under which the inhibition of the clotting process by the substrate is negligibleThe amidolytic activity is the sum of the activities of free thrombin and of the α2 macroglobulin-thrombin complex formed. Via a mathematical procedure the amount of SQ 68 that has been split by thrombin alone and not by the a2 macroglobulin-thrombin complex, can be derived from the course of the optical density.The total amount of SQ 68 eventually split by thrombin alone is proportional to the surface under the thrombin generation curve, i. e. to the time-integral of free thrombin. This value, that we call the thrombin potential (TP), directly indicates how much of any physiological substrate can potentially be split by the thrombin being generated in the plasma.

Summary.Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second-generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor V Leiden ), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC.Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non-OC users and in individuals that were heterozygous or homozygous for factor V Leiden . The response towards APC was evaluated on basis of the ratio (APC-sr) of the time integrals of thrombin formation determined in the presence and absence of APC.Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P < 0 . 001), independent of the kind of OC used. Women who used third-generation monophasic OC were significantly less sensitive to APC than women using second-generation OC (P < 0 . 001) and had APC-sr that did not significantly differ from heterozygous female carriers of factor V Leiden who did not use OC. Women who were heterozygous for factor V Leiden and used OC had APC-sr in the range of homozygous carriers of factor V Leiden . Two women who started OC therapy had significantly elevated APC-sr within 3 d.Acquired APC resistance may explain the epidemiological observation of increased risk for venous thrombosis in OC users, especially in women using third-generation OC.

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.