Received: 23 May 2019 | Accepted: 9 August 2019 DOI: 10.1111/jth.14612 ORIGINAL ARTICLE Polyphosphate, Zn2+ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting Yuqi Wang1 | Ivan Ivanov2 | Stephanie A. Smith1 | David Gailani2,3 | James H. Morrissey1,4 1Department of Biological Chemistry, University of Michigan Medical Abstract School, Ann Arbor, MI, USA Background: Inorganic polyphosphate modulates the contact pathway of blood 2 Department of Pathology, Microbiology & clotting, which is implicated in thrombosis and inflammation. Polyphosphate poly- Immunology, Vanderbilt University Medical Center, Nashville, TN, USA mer lengths are highly variable, with shorter polymers (approximately 60‐100 phos- 3Department of Medicine, Vanderbilt phates) secreted from human platelets, and longer polymers (up to thousands of University Medical Center, Nashville, TN, USA phosphates) in microbes. We previously reported that optimal triggering of clotting 4Department of Internal via the contact pathway requires very long polyphosphates, although the impact of Medicine, University of Michigan Medical shorter polyphosphate polymers on individual proteolytic reactions of the contact School, Ann Arbor, MI, USA pathway was not interrogated. Correspondence Objectives and methods: We conducted in vitro measurements of enzyme kinetics James H. Morrissey, Department of Biological Chemistry, 4301B MSRB III, 1150 to investigate the ability of varying polyphosphate sizes, together with high molecu- West Medical Center Drive, University of lar weight kininogen and Zn2+, to mediate four individual proteolytic reactions of the Michigan Medical School, Ann Arbor, MI 48109, USA. contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekal- Email: [email protected] likrein activation by factor XIIa, and prekallikrein autoactivation. Funding information Results: The individual contact pathway reactions were differentially dependent on National Heart, Lung, and Blood Institute, polyphosphate length. Very long‐chain polyphosphate was required to support fac- Grant/Award Number: R01 HL58837, R01 HL81326, R35 HL135823, R35 HL140025 tor XII autoactivation, whereas platelet‐size polyphosphate significantly accelerated and UM1 HL120877; American Heart the activation of factor XII by kallikrein, and the activation of prekallikrein by factor Association, Grant/Award Number: N026389 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. KEYWORDS blood coagulation factors, polyphosphates, prekallikrein, thrombosis, zinc Manuscript handled by: Alan Mast Final decision: Alan Mast J Thromb Haemost. 2019;17:2131–2140. wileyonlinelibrary.com/journal/jth © 2019 International Society on Thrombosis and | 2131 Haemostasis 2132 | WANG ET AL. 1 | INTRODUCTION Essentials The contact pathway of blood clotting—also known as the plasma • How individual contact pathway reactions depend on kallikrein‐kinin system—includes the circulating serine protease zy- polyphosphate length was not known. mogens, factor XII (FXII) and plasma prekallikrein (PK), as well as • Effect of polyphosphate, high‐MW kininogen and Zn2+ high‐molecular‐weight kininogen (HK), the non‐enzymatic cofactor on each reaction was investigated. for PK and a substrate for plasma kallikrein (PKa).1,2 This pathway, • The only reaction requiring very long‐chain polyphos- a schematic of which appears in Figure 1, is initiated when blood phate was factor XII autoactivation. or plasma is exposed to certain charged polymers or suitable sur- • Platelet‐size polyphosphate strongly promoted subsets faces.3 This interaction is thought to induce a conformational change of reactions of the contact pathway. in FXII that produces small amounts of enzymatically active FXIIa via autoactivation (more precisely, activation of FXII by FXIIa).4 FXIIa then cleaves PK to PKa, which can reciprocally activate more FXII to FXIIa.5 PK was also reported to undergo autoactivation to form PKa especially potent trigger of the contact pathway.18 In this study, we in the presence of a suitable polymer or surface.6 Once significant aimed to investigate the ability of various sizes of polyP, together concentrations of FXIIa accumulate, FXIIa activates factor XI (FXI) with two important cofactors, HK and Zn2+, to modulate four pro- to FXIa, triggering the intrinsic cascade of coagulation that leads to teolytic reactions of the contact pathway: FXII autoactivation, FXII thrombin generation and fibrin formation.7 The contact pathway is activation by PKa, PK activation by FXIIa, and PK autoactivation. also a critical connection for crosstalk between coagulation and in- PolyP is a highly negatively charged polymer of inorganic phos- flammation.2,8,9 However, this pathway is completely dispensable for phates that varies in length, depending on biological source. PolyP hemostasis, as individuals deficient in either FXII10,11 or PK12 have no released from platelets has a fairly narrow size range of approxi- bleeding tendencies. On the other hand, animal studies have shown mately 60‐100 phosphates long,19 whereas unicellular organisms that targeting FXII or PK is protective in thrombosis models,13-17 sug- contain heterogeneous polyP that can range up to thousands of gesting that the contact pathway could contribute to the develop- phosphates in length.20 Roles for polyP in prokaryotes and unicel- ment of thrombosis in humans. lular eukaryotes have been extensively studied21 but only recently While several artificial surfaces and polymers may activate have polyP's functions been explored in multicellular eukaryotes the contact pathway in vitro, the relevant (patho)physiologic acti- (particularly in mammals). Our previous work demonstrated that vators for this system in vivo have only recently been suggested.1 polyP acts at multiple stages of the clotting cascade, with pro‐hemo- Long‐chain polyphosphate (polyP), in particular, has emerged as an static, pro‐thrombotic, and pro‐inflammatory effects that are highly dependent on polyP chain length.18,22 In particular, we reported that optimal triggering of plasma clotting via the contact pathway 18 1 HK requires very long polyP polymers, although the polymer length dependence of each individual reaction of the contact pathway was FXI FXII FXIIa not investigated. In the present study we investigated the details of the requirement for very long‐chain polyP to trigger clotting via the 24 contact pathway. Individual enzyme reactions within the contact pathway can contribute to the complement cascade, fibrinolysis, PKa PK HK bradykinin generation, and inflammation.1,2 Accordingly, we hypoth- FXIa HK HK esized that the polyP length requirement might not be the same for 3 all four of the individual reactions in Figure 1, with platelet‐sized polyP possibly supporting subsets of these reactions. We now re- Intrinsic and common pathway port that only FXII autoactivation required very long‐chain polyP, whereas the reciprocal enzyme activation reactions (FXII activation Thrombin by kallikrein; prekallikrein activation by FXIIa) were accelerated to significant extents by platelet‐sized polyP. Furthermore, HK and Zn2+ are required only for specific subsets of these reactions. Blood clotting FIGURE 1 The following numbered reactions that contribute to initiation of the contact pathway of blood clotting were examined 2 | MATERIALS AND METHODS in this study: 1, activation of FXII by FXIIa (FXII autoactivation); 2, activation of FXII by PKa; 3, activation of PK by PKa (PK 2.1 | Materials autoactivation); and 4, activation of PK by FXIIa. Propagation of the clotting cascade happens when FXIIa activates FXI, leading Polybrene (hexadimethrine bromide), polyethylene glycol and soy- ultimately to thrombin generation bean trypsin inhibitor were from Sigma. Human FXII, α‐FXIIa, PK, WANG ET AL. | 2133 PKa, and single‐chain HK were from Enzyme Research Laboratories. PKa, 100 nmol/L HK, and 10 μmol/L ZnCl2, in the presence of 0 to CTI was from Haematologic Technologies (Essex Junction). 100 μmol/L polyP1200 or polyP79. Experiments evaluating the polyP Polyclonal goat anti‐human PK antibody (IgG) was from Affinity size‐dependence contained 100 nmol/L HK and 10 μmol/L ZnCl2, Biologicals. The chromogenic substrate for FXIIa or PKa, H‐D‐Pro‐ in the absence or presence of 10 μmol/L narrowly size‐fraction- Phe‐Arg‐pNA (L‐2120), was from Bachem. Chelex® 100 Resin was ated polyP. Experiments evaluating the impact of ZnCl2 included from Bio‐Rad Laboratories. Ultra‐Low Attachment microplates were 10 μmol/L polyP1200, 100 nmol/L HK, and either 2 mmol/L EDTA from Corning Inc. or 0 to 20 μmol/L ZnCl2. Experiments evaluating the impact of HK Two preparations of heterogeneous, long‐chain polyP employed included 10 μmol/L polyP1200, 10 μmol/L ZnCl2, and 0 to 900 nmol/L in this study were prepared starting from chemically synthesized, HK. Timed aliquots (10 μL) were removed and quenched in 70 μL ice‐ high molecular weight polyP (Sigma‐Aldrich). One preparation,