Activity of factor XII‐Locarno

Abstract Essentials Conversion of FXII to α‐FXIIa on surfaces requires cleavage after Arginine 353. Replacing Arg353 with alanine results in a single chain form (FXII‐R353A) that has some activity. Replacing Arg‐353 with proline (FXII‐Locarno, FXII‐R353P) reduces activity of single chain FXII compared to FXII‐R353A. Proper conformation of the FXII activation loop is required for single chain FXII activity. Background Factor XII (FXII) Locarno is a natural variant with proline replacing Arg353 at the activation cleavage site, preventing conversion to the fully active protease factor XIIa (FXIIa). Recently, we showed that FXII restricted to a single chain form (sc‐FXII) by replacing Arg353 with alanine expresses proteolytic activity that is enhanced by cofactors such as polyphosphate. Objective To determine if the Pro353 substitution affects the activity of sc‐FXII. Methods Wild type FXII (FXII‐WT), FXII‐R353A, and FXII Locarno (FXII‐R353P) were tested for their abilities to activate prekallikrein, and to induce thrombin generation and coagulation in plasma in a factor XI‐dependent manner. Results FXII‐WT is converted to FXIIa by autoactivation in the presence of polyphosphate, and by incubation with kallikrein. FXII‐R353P and FXII‐R353A were not converted to FXIIa by these methods. Despite this, FXII‐R353A converts prekallikrein to kallikrein, and the reaction is enhanced by polyphosphate. FXII‐R353P also converts prekallikrein to kallikrein, but at a slower rate than FXII‐R353A. In FXII‐deficient plasma induced to clot with silica, FXII‐R353A is a better promoter of factor XI‐dependent thrombin generation and coagulation than FXII‐R353P. Conclusion The activity of sc‐FXII is sensitive to perturbations in the activation loop, which contains residue 353. Homology modeling based on the crystal structure of the FXII homolog tissue plasminogen activator suggests that Pro353 introduces changes in the shape and flexibility of the activation loop that disrupt key interactions that support an active conformation in sc‐FXII.


| INTRODUCTION
Exposure of blood to a variety of biological and non-biological substances or surfaces initiates contact activation. Central to this process is the plasma protein factor XII (FXII), the precursor of the serine protease α-factor XIIa (α-FXIIa). 1 In blood plasma, α-FXIIa activates the kallikreinkinin system by converting plasma prekallikrein (PK) to α-kallikrein, 2 and initiates coagulation by activating factor XI (FXI). 3 It is well-recognized that FXII undergoes autocatalysis to α-FXIIa by cleavage after Arg353 upon surface binding, 4 however, the mechanism that initiates this process has been debated. Recently, we showed that FXII restricted to the single-chain precursor form (sc-FXII) by replacement of Arg353 with alanine still expresses proteolytic activity toward FXII, PK, and FXI in the presence of anionic cofactors such as polyphosphate (Poly-P). 5 While the activity of sc-FXII is orders of magnitude lower than that of α-FXIIa, it would provide a triggering mechanism for contact activation.
FXII-Locarno is a cross-reactive material positive FXII variant that contains proline in place of Arg353 (FXII-R353P). It was identified in a patient with a prolonged activated partial thromboplastin time (aPTT) and no bleeding symptoms. 6,7 Like FXII-R353A, FXII-R353P cannot be converted to α-FXIIa, and was initially thought to lack proteolytic activity. 6,7 We compared the activities of FXII containing FXII-R353A and FXII-R353P, to determine if the non-conservative proline substitution at the activation cleavage site altered the activity of sc-FXII.

| Recombinant proteins
were prepared by site-directed mutagenesis.
At various times, aliquots were removed into reducing SDS-sample buffer, size fractionated by SDS-PAGE and evaluated by western blot using goat anti-human FXIl IgG. Detection was with HRP conjugated rabbit anti-goat IgG and chemiluminescence.
Thrombin generation was initiated by adding 10 μL of 20 mmol L −1 HEPES pH 7.4, 100 mmol L −1 CaCl 2 , 6% BSA and fluorescence was monitored over 60 min on a Fluoroskan Ascent fluorometer, and converted to thrombin generated using the manufacturers software.
Assays were performed in triplicate. Endogenous thrombin potential (ETP-area under the curve, reported in nm.min) was determined using GraphPad Prism software.

| Plasma clotting assays
aPTT assays were performed on a STart4 Coagulation Analyzer (Diagnostica Stago). PNP or FXII-dp supplemented with vehicle or FXII were tested, using PTT-A reagent to trigger contact activation. Assays were performed in triplicate.
When FXII and PK are mixed in buffer, reciprocal conversion to α-FXIIa and α-kallikrein occurs by a reaction enhanced by Poly-P (Figure 2, left). 5,10 PK undergoes little autocatalysis in the presence of Poly-P ( Figure 1C), 5 and is dependent on FXII to convert it to α-kallikrein. As reported, FXII-R353A catalyzes PK conversion to α-kallikrein ( Figure 2, middle), 5 demonstrating the proteolytic activity of sc-FXII. FXII-R353P also catalyzes PK activation, but at a lower rate than FXII-R353A (Figure 2, right), suggesting structural changes introduced by Pro353 compromise activity. PK circulates as a complex with the cofactor HK, which enhances FXIIa-mediated PK activation on surfaces. 16 HK did not enhance PK activation by FXII-R353A or FXII-R353P with Poly-P (data not shown).
α-FXIIa promotes thrombin generation in plasma by activating FXI, a homolog of PK. 3 We assessed the capacity of FXII to activate FXI using thrombin generation assays. Little thrombin generation occurs when FXII species are added to FXII-dp in the absence of an inducer of contact activation ( Figure 3A, left). The small peak produced by FXII-WT probably reflects a trace of FXIIa in the FXII-WT preparation. Adding a silica-based reagent to plasma containing FXII-WT promotes robust thrombin generation ( Figure 3A, middle, peak 499 ± 2 nmol L −1 ; ETP 1909 ± 4 nmol L −1 min −1 ). FXII-R353A also supports surface-induced thrombin generation, but with reduced activity compared to FXII-WT (peak 82 ± 4 nmol L −1 ; ETP 559 ± 19 nm min −1 ). The difference between FXII-WT and FXII-R353A likely reflects conversion of the former to α-FXIIa.
FXII-R353P had a smaller effect on thrombin generation than FXII-R353A (peak 15 ± 1 nmol L −1 ; ETP 143 ± 2 nmol L −1 min −1 ) that was We compared FXI-WT, FXII-R353A, FXII-R353P in aPTT assays, which also require FXI activation by contact activation. In Figure 3B, aPTTs of PNP (36.4 ± 2.1 seconds) and FXII-dp (271 ± 5.1 seconds) are shown as white bars, while yellow bars show the effects of FXII-WT on the aPTT of FXII-dp, up to the normal plasma FXII concentration (400 nmol L −1 ). FXII-R353A and FXII-R353P shorten the aPTT of FXII-dp slightly, with effects equivalent to <1% of the activity of FXII-WT. This supports the premise that it is α-FXIIa that is primarily responsible for FXI activation in normal plasma in the aPTT. The effect of FXII-R353A was significantly greater than FXII-R353P (206.2 ± 6.1 vs 256.5 ± 3.3, respectively, P < .05). Taken as a whole, the data with PK activation and thrombin generation assays, and the aPTT data, suggest that proline at position 353 in FXII has a deleterious effect on sc-FXII activity. Activation of serine protease zymogens typically involves internal proteolysis within an activation loop, creating a free N-terminus for the catalytic domain that stabilizes the protease S1 specificity pocket. In human FXII this cleavage is after Arg353. Recently, we showed that forms of FXII that are not cleaved after Arg353 (including FXII-R353A) still activate their natural substrates PK, FXI and FXII in the presence of anions such as polyphosphate. The rates of activation for sc-FXII-mediated reactions are orders of magnitude lower than for FXIIa-mediated reactions, implying that the biological activities of FXII are mediated primarily through α-FXIIa. We suspect that the activity intrinsic to sc-FXII serves as a trigger for surfacedependent conversion of FXII to α-FXIIa, with α-FXIIa then producing physiologic effects.
Our data show that FXII-R353P has reduced activity compared to FXII-R353A, indicating sc-FXII activity is sensitive to changes in the conformation of the activation loop on which residue 353 resides.
The FXII catalytic domain is homologous to that of tissue plasminogen activator (tPA), a protease that expresses substantial activity in its single-chain form (sc-tPA). 17,18 To compare FXII and tPA, we use the numbering system for chymotrypsin in which the activation cleavage site arginine (Arg353 in FXII, Arg275 in tPA,) is designated residue 15. In most trypsin-like proteases the activation loop is relatively unstructured. However, in the sc-tPa structure the activation loop is well-defined, forming interactions with components of the catalytic domain around the S1 pocket. 18 Figure 4A shows these interactions, with the activation loop (light blue) packing against the 180-loop (green) and 140-loop (red). Residues Ile16 to Gly18 form a β-turn, with Gly18 forming a hydrogen bond with Asp189. Lys156 at the 140-loop C-terminus forms a salt bridge with Asp194 that is thought to stabilize the S1 pocket. 18 Figure 4B shows a homology model based on the sc-tPA structure that suggests similar interactions in sc-FXII. Val16-Gly18 forms a β-turn in the activation loop. FXII Gln156 would not form a salt bridge with Asp194 in the same manner as Lys156 in sc-tPA, but could form a hydrogen bond that provides some stability to the S1 pocket. The bulky Pro353 in FXII Locarno is predicted to induce curvature in the main chain due to preference for discrete torsion angles

RELATIONSHIP DISCLOSURE
None of the authors have any disclosures relevant to this paper.