Polyphosphate, Zn2+ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting

Abstract

Background: Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60–100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. Objectives and methods: We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+, to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. Results: The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. Conclusions: Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI

“…PKa 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 20% 1,2dioleoylsn-glycero-3-phosphoethanolamine (DOPE), and 20% 1,2dioleoyl-sn-glycero-3-phosphoserine (DOPS), and, further, that the presence of HK does not have any effect on the reaction. In a study of the activation of FXII by PKa, Wang et al (34) reported a requirement of HK for PKa; however, our data suggest that HK has little effect on the FIX-activating activity of PKa. In summary, our data show that the activation of FIX by PKa can happen in the absence of a surface or in the presence of PLs, and that HK plays no role as an accelerating protein cofactor under these conditions.…”

Proteolytic activity of contact factor zymogens

“…During surface‐induced contact activation, FXIIa catalyzes (a) rapid conversion of PK to PKa, (b) conversion of FXII to FXIIa (autoactivation), and (c) conversion of FXI to FXIa 3‐5,30,31 . In the following experiments orthophosphate polymers (polyphosphate) were used as a contact surface 10,11,51 . Specifically, we used relatively short chain polyphosphates containing 60 to 100 phosphate units, which are similar to those in platelet dense granules that are released upon platelet activation 10,51 .…”

“…[3][4][5]30,31 In the following experiments orthophosphate polymers (polyphosphate) were used as a contact surface. 10,11,51 Specifically, we used relatively short chain polyphosphates containing 60 to 100 phosphate units, which are similar to those in platelet dense granules that are released upon platelet activation. 10,51 Polyphosphate accelerated PK activation by FXII-T ( Figure 4E), 14 indicating surface binding alters the conformation of PK, FXII-T, or both to facilitate PK conversion to PKa.…”

“…Coagulation through this pathway starts when plasma is exposed to a “surface.” 3‐5 A variety of inorganic and organic substances, many of which carry a net negative charge, function as pro‐coagulant surfaces (Figure 1B). 3‐11 FXII undergoes autocatalytic conversion to FXIIa in the presence of such substances (Figure 1C), 12‐14 suggesting a triggering mechanism for the cascade. However, the question of how autocatalytic activity arises from an inactive state has been a point of controversy.…”

“…of which carry a net negative charge, function as pro-coagulant surfaces ( Figure 1B). [3][4][5][6][7][8][9][10][11] FXII undergoes autocatalytic conversion to FXIIa in the presence of such substances ( Figure 1C), [12][13][14] suggesting a triggering mechanism for the cascade. However, the question of how autocatalytic activity arises from an inactive state has been a point of controversy.…”