SummaryRecently a new hemostatic disorder has been described which appears to be an important risk factor for familial thromboembolism. The disorder is characterized by a poor anticoagulant response to activated Protein C (APC) and has been shown to be due to lack of an APC cofactor activity which is a property of factor V.A kit for determining the response of plasma samples towards addition of APC in an APTT-based assay - COATEST APC Resistance -has been evaluated on 35 coagulation instruments in a multicenter study involving 32 laboratories. A lyophilized normal plasma and identical plasma aliquots from 20 individuals, one of whom had a borderline resistance to APC, were analysed in each laboratory and the sensitivity of each plasma to APC was determined as the ratio between the clotting times obtained in the presence and absence of APC (APC ratio).The plasma from the individual with a borderline resistance to APC activity was correctly classified as the lowest responder in each laboratory, with an APC ratio in the range 1.6-2.4. In comparison, plasmas from individuals with a pronounced response to APC activity resulted in APC ratios above 3.4 in most cases. Interestingly, although the actual APT time for a plasma from a given individual showed a more than 10 s difference due to the type of instrumentation used, the variation in the APC ratio was limited. A similar discrimination was also obtained from evaluation of the actual prolongation of the clotting time in the presence of APC.The intra-laboratory coefficient of variation for the clotting times were on average 2.0% and 3.9% in the absence and presence of APC, respectively, indicating that the precision for the prolonged clotting times obtained in the presence of APC is sufficient to allow a safe assignment of the APC response. The APC ratio for the lyophilized normal plasma was 2.7 ± 0.2 (2 S.D.) illustrating a narrow distribution between instruments which shows the feasibility of including such plasma for assay validation. Altogether, the results indicate that all the coagulation instruments included in the study can be used for detection of individuals with resistance to APC activity through determination of the APC ratio or the prolongation time.

BackgroundFibrinogen and prothrombin have been suggested to become rate limiting in trauma associated coagulopathy. Administration of fibrinogen is now recommended, however, the importance of prothrombin to patient outcome is unknown.MethodsWe have utilized two trauma patient databases (database 1 n = 358 and database 2 n = 331) to investigate the relationship of plasma prothrombin concentration on clinical outcome and coagulation status. Database 1 has been used to assess the relationship of plasma prothrombin to administered packed red blood cells (PRBC), clinical outcome and coagulation biomarkers (Prothrombin Time (PT), ROTEM EXTEM Coagulation Time (CT) and Maximum Clot Firmness (MCF)). ROC analyses have been performed to investigate the ability of admission coagulation biomarkers to predict low prothrombin concentration (database 1), massive transfusion and 24 h mortality (database 1 and 2). The importance of prothrombin was further investigated in vitro by PT and ROTEM assays in the presence of a prothrombin neutralizing monoclonal antibody and following step-wise dilution.ResultsPatients who survived the first 24 h had higher admission prothrombin levels compared to those who died (94 vs.67 IU/dL). Patients with lower transfusion requirements within the first 24 h (≤10 units of PRBCs) also had higher admission prothrombin levels compared to patients with massive transfusion demands (>10 units of PRBCs) (95 vs.62 IU/dL). Admission PT, in comparison to admission ROTEM EXTEM CT and MCF, was found to be a better predictor of prothrombin concentration <60 IU/dL (AUC 0.94 in database 1), of massive transfusion (AUC 0.92 and 0.81 in database 1 and 2 respectively) and 24 h mortality (AUC 0.90 and 0.78 in database 1 and 2, respectively). In vitro experiments supported a critical role for prothrombin in coagulation and demonstrated that PT and ROTEM EXTEM CT are sensitive methods to measure low prothrombin concentration.DiscussionOur analyses suggest that prothrombin concentration at admission is predictive of mortality and transfusion and indicates that prothrombin and fibrinogen are rate limiting in coagulopathy.ConclusionsAdmission PT is predictive of low prothrombin concentration and clinical outcome. PT could therefore be used as a surrogate for prothrombin concentration and further evaluation of point-of-care devices for faster PT analysis is warranted.

AZD0837, currently in clinical development, is a once-daily oral anticoagulant that is bioconverted to AR-H067637, a selective, reversible direct thrombin inhibitor (DTI). When developing a new DTI, the antithrombotic effects are commonly investigated in in vivo animal models; this report shows the effect of AR-H067637 in venous and arterial thrombosis and bleeding models in anaesthetised rats. Thrombus formation was induced by topical application of ferric chloride to the carotid artery or to the caval vein with partial stasis. Cutaneous incision bleeding time and muscle transection blood loss were assessed, with or without acetylsalicylic acid (ASA). Activated partial thromboplastin time (APTT), ecarin coagulation time (ECT) and thrombin coagulation time (TCT) were used as plasma biomarkers of anticoagulant effect. Dalteparin was used as a reference compound. AR-H067637, given by continuous infusion, displayed a dose-dependent antithrombotic effect, with 50% inhibition (IC50) of thrombus size in venous and arterial thrombosis models obtained at plasma concentrations of 0.13 μM and 0.55 μM, respectively, without increased bleeding. Dose-dependent increased bleeding and blood loss were seen at plasma concentrations ≥1 μM AR-H067637. At the highest AR-H067637 plasma concentration tested, bleeding time and blood loss increased two and four times the vehicle group. Addition of ASA moderately potentiated bleeding time and blood loss. APTT, ECT and TCT were dose-dependently prolonged. These studies demonstrate that the DTI AR-H067637 inhibits thrombus formation in rat venous and arterial thrombosis models with no or minor increases in bleeding.

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