A factor XIa-activatable hirudin-albumin fusion protein reduces thrombosis in mice without promoting blood loss.

BACKGROUND: Hirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects. Because hirudin's N-terminus must engage thrombin's active site for effective inhibition, fusing a cleavable peptide at this site may improve hirudin's risk/benefit ratio as a therapeutic agent. Previously we engineered a plasmin cleavage site (C) between human serum albumin (HSA) and hirudin variant 3 (HV3) in fusion protein HSACHV3. Because coagulation factor XI (FXI) is more involved in thrombosis than hemostasis, we hypothesized that making HV3 activity FXIa-dependent would also improve HV3's potential therapeutic profile. We combined albumin fusion for half-life extension of hirudin with positioning of an FXIa cleavage site N-terminal to HV3, and assessed in vitro and in vivo properties of this novel protein. RESULTS: FXIa cleavage site EPR was employed. Fusion protein EPR-HV3HSA but not HSAEPR-HV3 was activated by FXIa in vitro. FVIIa, FXa, FXIIa, or plasmin failed to activate EPR-HV3HSA. FXIa-cleavable EPR-HV3HSA reduced the time to occlusion of ferric chloride-treated murine arteries and reduced fibrin deposition in murine endotoxemia; noncleavable mycHV3HSA was without effect. EPR-HV3HSA elicited less blood loss than constitutively active HV3HSA in murine liver laceration or tail transection but extended bleeding time to the same extent. EPR-HV3HSA was partially activated in citrated human or murine plasma to a greater extent than HSACHV3. CONCLUSIONS: Releasing the N-terminal block to HV3 activity using FXIa was an effective way to limit hirudin's bleeding side-effects, but plasma instability of the exposed EPR blocking peptide rendered it less useful than previously described plasmin-activatable HSACHV3.

Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood coagulation.

The process of tissue factor initiated blood coagulation is discussed. Reactions of the blood coagulation cascade are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are assembled on a membrane surface in a calcium-dependent manner. These complexes are 105-109-fold more efficient in proteolyses of their natural substrates than enzymes alone. Based upon data acquired using several in vitro models of blood coagulation, tissue factor initiated thrombin generation can be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the generation of nanomolar amounts of thrombin, femto- to picomolar amounts of factors VIIa, IXa, Xa, and XIa, partial activation of platelets, and almost quantitative activation of procofactors, factors V and VIII. The duration of this phase is primarily influenced by concentrations of tissue factor and TFPI. The characteristic features of the propagation phase are: almost quantitative prothrombin activation at a high rate, completion of platelet activation, and solid clot formation. This phase is primarily regulated by antithrombin III and the protein C system. Thrombin generation during the propagation phase is remarkably suppressed in the absence of factor VIII and IX (hemophilia A and B, respectively) and at platelet counts <5% of mean plasma concentration. The majority of data accumulated in in vitro models and discussed in this review are in good agreement with the results of in vivo observations.

Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia.

Evidence of factor XI (FXI) activation in vivo is scarce. In addition, it remains uncertain whether thrombin, factor XIIa (FXIIa), or perhaps another protease is responsible for FXI conversion. We investigated the activation of FXI in eight healthy volunteers after infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI activation was measured with a novel enzyme capture assay that detects noncomplexed FXIa. Activation of FXI was apparent with a significant plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of 11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with previous studies, thrombin generation was detected 1 hour after endotoxin infusion to become maximal after 3 to 4 hours. In contrast, we did not find any evidence of contact activation, because markers of activation of prekallikrein and FXII remained undetectable. From the FXIa data a theoretical model was constructed which suggested that inhibition of FXIa does not take place in the plasma compartment, but is localized on a surface. These data provide the first evidence for FXI activation in low-grade endotoxemia and suggest that FXI is activated independently of FXII.