ALBERT

Description: Key Points FXI must be a dimer for normal activation by fXIIa but not for activation by thrombin or autoactivation. Poly-P is a cofactor for activation of coagulation fXI by fXIIa and thrombin and supports fXI autoactivation.

Description: Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

Description: Key PointsThe single-chain form of FXII, a component of the plasma contact system, has proteolytic activity. Single-chain FXII activity suggests a mechanism of contact activation initiation when blood is exposed to physiologic/artificial surfaces.

Description: Abstract 2235 Conversion of factor IX (fIX) to the protease factor IXaβ (fIXaβ) is an important reaction during thrombin generation at a site of vascular injury. The physiologic activators of fIX are the proteases factor VIIa and factor XIa (fXIa). The zymogen of fXIa, fXI, is a 160 kDa dimer of two identical subunits linked by a disulfide bond. Each subunit has four apple domains at the N terminus (A1-A4), and a trypsin-like catalytic domain at the C-terminus. Conversion of fXI to fXIa involves cleavage of each subunit at the Arg369-Ile370 bond, generating a heavy chain (the apple domains) and an activated catalytic domain that remains connected to the heavy chain by a disulfide bond. FXIa activates fIX in the presence of calcium ions by sequential cleavage after Arg145 (forming the inactive intermediate fIXα) and then after Arg180 to form fIXaβ. Previously, we showed that an exosite (a site on fXIa distinct from the active site) on the A3 domain of the fXIa heavy chain is a major determinant of affinity and specificity for fIX activation by fXIa (J Biol Chem 1999;274:36373 and 2005;280:23523). Evidence has also been presented for a second fIX-binding exosite on the fXIa catalytic domain. While the catalytic efficiency (kcat/Km) for fIX activation by an isolated fXIa catalytic domain (fXIaCD – no heavy chain) was ∼500 fold lower than activation by fXIa, this was reported to be due to a decrease in kcat, rather than the expected increase in Km that should accompany loss of the A3 exosite (Biochemistry 2007;46:9830). To investigate this discrepancy, we used recombinant wild type fXIa (fXIaWT), fXIa missing the exosite on the A3 domain (fXIa-PKA3) or fXIaCD to activate purified fIX and fIXα. Full progress curves were generated using densitometry of Coomassie Blue stained SDS-polyacryalmide gels imaged at infrared wavelengths. The Km and kcat for cleavage by fXIaWT of fIX after Arg145 (Km 0.09 ± 0.02 μM, kcat = 7.3 ± 0.4 min−1) and fIXα after Arg180 (Km 0.12 ± 0.02 μM, kcat = 6.8 ± 0.4 min−1) are similar, and agree with published results. FXIa/PKA3 cleaved fIX after Arg145 with a significantly higher Km (〉2 μM), consistent with loss of the exosite, and leading to an ∼100-fold reduction in catalytic efficiency. Because we were not able to reach saturation, it is not clear if the kcat was affected appreciably. Catalytic efficiency for cleavage after Arg180 was ∼3000-fold lower with FXIa-PKA3 than with fXIaWT, but the slow rate of cleavage precluded clearly determining if this was due to an effect on Km or kcat. These results indicate that the A3 exosite is involved in both cleavages, and loss of the exosite has a more deleterious effect on the second cleavage after Arg180 that converts fIXα to fIXaβ than the first cleavage after Arg145 that converts fIX to fIXα. This would account for the observation that there is substantial accumulation of fIXα when fIX is activated by FXIa-PKA3, but not by fXIaWT. For fIX cleavage after Arg145 by fXIaCD, Km was again markedly increased (≥ 2 μM) compared to FXIaWT, with a modest (∼3-fold) reduction in kcat resulting in reduced catalytic efficiency that is roughly similar to that for FXIa/PKA3. The catalytic efficiency of cleavage after Arg180 by fXIaCD was ∼4000 fold reduced compared to FXIa-WT. Interestingly, when calcium was removed from the reactions, cleavage of both the Arg145 and Arg180 activation sites by fXIa-WT, but not by fXIa/PKA3 or fXIaCD, were markedly impaired, indicating both cleavages are Ca2+–dependent reactions. Cumulatively, these results indicate that an exosite on the heavy chain A3 domain is largely responsible for the Ca2+-dependent affinity of fIX and fIXα for fXIa. We used surface plasmon resonance as a complementary approach to look directly at Ca2+-dependent binding of fXIa to fIX. FXIa-WT bound to immobilized fIX with Kd 48nM, in reasonable agreement with results from the kinetic analysis. Isolated fXIa heavy chain (lacking the catalytic domain) bound with similar Kd (53 nM). In contrast, fXIa/PKA3 and fXIaCD bound poorly to fIX (Kd 〉2 μM). Taken as a whole, the data support the hypothesis that an exosite on the fXIa A3 domain is largely responsible for affinity and specificity of the fXIa-mediated reactions converting fIX to fIXα, and fIXα to fIXaβ. While the analysis cannot rule out minor contributions of other exosites to the reactions, they do not support the premise that there is a fIX- or fIXα-binding site on the fXIa catalytic domain that contributes substantially to initial substrate binding. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2244 In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV. We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not. In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX. Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures: Tucker: Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Description: The prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to β-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by β-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, β-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

Description: Abstract 1150 During plasma coagulation the protease α-thrombin (αIIa) cleaves fibrinogen to form a fibrin clot. Conversion of prothrombin to αIIa is catalyzed by factor (f) Xa, and results in expression of two electropositive regions on αIIa designated anion binding exosites (ABE) I and II. ABE I is involved in fibrinogen binding. In the presence of fVa and phospholipid, fXa cleaves prothrombin preferentially after Arg320, generating the intermediate meizothrombin (MzIIa), which also expresses ABE I. MzIIa is rapidly converted to αIIa. αIIa can be converted to β-thrombin (βIIa) and γ-thrombin (γIIa), both of which are cleaved within ABE I, and have greatly reduced capacity to convert fibrinogen to fibrin. Physiologic functions for βIIa or γIIa are not established; however, both have been identified in clotting blood. αIIa up-regulates its own generation in plasma by converting fXI to the protease fXIa. Yun et al. (J Biol Chem 2003;278:48112) showed that amino acids in ABE I are required for optimal fXI activation in the presence of the polyanion dextran sulfate (DS). MzIIa also activates fXI, consistent with a role for ABE I in protease binding to fXI. Given the absence of ABE I in βIIa and γIIa, it seems reasonable to postulate these proteases would interact poorly with fXI. In a clotting assay in which thrombin is added to plasma anticoagulated with citrate (low calcium), βIIa (12.5 nM) and γIIa (50 nM) did not induce clot formation, consistent with their low capacity to cleave fibrinogen. However, when plasma was recalcified to allow thrombin to form from endogenous prothrombin, both βIIa and γIIa induced clot formation. Recalcified plasma in the absence of βIIa or γIIa did not clot (800 sec observation period), indicating fibrin formation was βIIa/γIIa-dependent. Addition of an antibody to fXI prolonged the clotting time with βIIa, and prevented clotting with γIIa, suggesting βIIa and γIIa were activating fXI. In addition, with γIIa, a fXIIa inhibitor modestly prolonged clotting time, indicating the plasma contact phase was activated. We studied fXI activation by thrombin using western blot. βIIa and γIIa activated fXI at approximately half the rate of αIIa, while MzIIa activated fXI ∼4 fold faster than αIIa. FXI activation by αIIa is greatly enhanced by DS. In the presence of DS, αIIa and βIIa activated fXI comparably, while results with γIIa were not informative because the protease does not interact well with DS. Importantly, fXI activation by αIIa was not affected by the ABE I blocking peptide hirugen, indicating ABE I is not required for fXI activation by thrombin. While βIIa and γIIa were less effective fXI activators than αIIa and MzIIa in solution, significantly different results were obtained in a plasma thrombin generation assay. Here coagulation is initiated in fXII deficient plasma with thrombin (10 nM), and subsequent thrombin generation from endogenous prothrombin is monitored. The system is fXI-dependent, as a fXI antibody blocks thrombin generation. Prior work with this system indicates fXI is probably converted to fXIa by the thrombin added to initiate the process. Initiation of coagulation with αIIa and MzIIa resulted in comparable thrombin generation (∼250 nM). βIIa and γIIa, as well as recombinant αIIa with mutations in amino acids in ABE I induced thrombin generation ∼2-fold greater than for αIIa and MzIIa. We hypothesized this was due to the inability of fibrinogen to compete with fXI for binding to thrombin species lacking ABE I. Consistent with this, hirugen peptide enhanced αIIa initiated thrombin generation ∼4-fold. Finally, we followed up on the observation that a fXIIa inhibitor prolonged time to γIIa-induced clot formation in recalcified plasma. In solution, γIIa, but not αIIa, βIIa, or MzIIa cleaves the contact factors fXII and PK. The cleaved proteases, in turn, are capable of cleaving chromogenic substrates, and have activity in a reciprocal fXII-PK activation assay. Our studies show that ABE I is not required for thrombin-mediated activation of fXI, that thrombin species not fully expressing ABE I may be better than αIIa and MzIIa as initiators of fXI-dependent thrombin generation in plasma, and that γIIa can activate the plasma contact proteases. Taken as a whole, the data indicate forms of thrombin other than αIIa may contribute directly to feedback activation of fXI, and may represent a previously unrecognized link between coagulation and the contact system. Disclosures: No relevant conflicts of interest to declare.

Description: The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.