Summary Haemophilia A carriers have historically been thought to demonstrate normal haemostasis. However, recent data demonstrates that despite normal factor VIII, haemophilia A carriers demonstrate an increased bleeding tendency. We tested the hypothesis that obligate haemophilia carriers demonstrate an increase in bleeding symptoms. A cross sectional study was performed comparing haemophilia A carriers to normal women. Questionnaire assessment included a general bleeding questionnaire, condensed MCMDM-1VWD bleeding assessment tool and Pictorial Bleeding Assessment Chart (PBAC). Laboratory assessment included complete blood count, prothrombin time, activated partial thromboplastin time, fibrinogen activity, FVIII activity (FVIII:C), von Willebrand factor antigen level, ristocetin cofactor, platelet function analyser-100™ and ABO blood type. 44 haemophilia A carriers and 43 controls were included. Demographic features were similar. Laboratory results demonstrated a statistically significant difference only in FVIII:C (82.5 versus 134%, p value < 0.001). Carriers reported a higher number of bleeding events, and both condensed MCMDM-1 VWD bleeding scores (5 versus 1, p value < 0.001) and PBAC scores (423 versus 182.5, p value = 0.018) were significantly higher in carriers. Haemophilia A carriers exhibit increased bleeding symptoms when compared to normal women. Further studies are necessary to fully understand the bleeding phenotype in this population and optimize clinical management.

• Activated factor XI binds and proteolyzes tissue factor pathway inhibitor.• Activated factor XI promotes factor X activation generation and fibrin formation through the inactivation of tissue factor pathway inhibitor from platelets and on endothelial cells.Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium-and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI-or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI.

Summary. Background: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. Methods: We enrolled 17 patients undergoing firsttime cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzymelinked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. Results: The ELISA and Western blot data indicated that an increase in fulllength TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the fulllength TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. Conclusions: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.

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