Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation

Abstract

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

Single-chain factor XII: a new form of activated factor XII

“…While coagulation through the intrinsic pathway begins with fXII activation, the mechanism that initiates the process has been uncertain. The data presented here suggest that fXII expresses sufficient proteolytic activity in its single-chain form to initiate surface-dependent contact activation [69**]. Given the low level of activity relative to α-fXIIa, sc-fXII activity is probably most important early in contact activation.…”

“…The consequences of cleavage after Arg343 are not clear. To test the hypothesis that sc-fXII is a protease, we replaced Arg334, Arg343, and Arg353 with alanine [69**]. The resulting protein, designated fXII-T (for “triple” mutant), does not undergo autocatalysis to α-fXIIa in the presence of polyphosphate, and is resistant to cleavage by α-kal and fXIIa.…”

“…When wild type fXII (fXII-WT) is mixed with PPK at plasma concentrations there is reciprocal conversion of the proteins to α-fXIIa and α-kal (Figure 4A) [69**,70]. Initiation of this process is often attributed to traces of α-fXIIa in fXII preparations.…”

“…Initiation of this process is often attributed to traces of α-fXIIa in fXII preparations. However, when fXII-WT is replaced by fXII-T, PPK is still converted to α-kal, although at a lower rate than with fXII-WT [69**]. PPK activation by fXII-T turns out to be four orders of magnitude slower than with α-fXIIa.…”

Contact ignition by single-chain XIIa

“…n In this issue of Blood, Gluckman et al confirm the role of HLA identical sibling transplantation in sickle cell disease (SCD) with a 5-year overall survival of 95% when performed at an early age (,16 years) and using bone marrow as the source of stem cells. 1 A lthough SCD is the most common inherited hemoglobinopathy worldwide, and most patients suffer from life-threatening complications, fewer transplants have been performed compared with the number carried out for thalassemia major. Past indications for stem cell transplantation (SCT) reserved this procedure for patients with significant sicklerelated morbidity, such as recurrent pain crises leading to multiple hospitalizations, stroke or acute chest syndrome, sickle lung disease, and sickle nephropathy.…”