Polyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (> 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (ϳ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required > 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens. IntroductionPolyphosphate (polyP)-a linear polymer of inorganic phosphateaccumulates in a variety of microorganisms 1 and is secreted by activated human platelets. 2,3 We recently showed that polyP is a potent modulator of the human blood-clotting system. [3][4][5][6] The polymer lengths of polyP are known to vary substantially among different organisms and cell types, with relatively short polymers being secreted by human platelets (ϳ 60-100 phosphate units long) 2,3 and very long polymers accumulating in microorganisms (many hundreds to more than 1000 phosphate units long). 1 In this study, we demonstrate that shorter versus longer polymers of polyP have differential effects on the blood clotting system, with important physiologic/pathophysiologic implications. PolyP has been widely described in unicellular organisms such as bacteria, fungi, algae, and protozoa, where it plays diverse physiologic roles, including regulating growth, stress responses, and virulence. 1,7 Comparatively less is known about the metabolism or physiologic roles of polyP in mammalian cells, 8 although polyP is reported to induce apoptosis in plasma cells, 9 promote calcification in osteoblasts, 10 block metastasis of melanoma cells in a mouse model, 11 and possibly serve as a regulatory factor in proliferative signaling pathways. 12 PolyP is present at high concentrations in dense granules of human platelets and is secreted upon platelet activation. 2,3 PolyP has a half-life in plasma of approximately 90 minutes, because of degradation by phosphatases. 4,13 We recently showed that polyP is a potent hemostatic regulator, acting at 3 points in the blood clotting cascade: it...

AbstractInorganic polyphosphate is widespread in biology and exhibits striking prohemostatic, prothrombotic, and proinflammatory effects in vivo. Long-chain polyphosphate (of the size present in infectious microorganisms) is a potent, natural pathophysiologic activator of the contact pathway of blood clotting. Medium-chain polyphosphate (of the size secreted from activated human platelets) accelerates factor V activation, completely abrogates the anticoagulant function of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. Polyphosphate may have utility as a hemostatic agent, whereas antagonists of polyphosphate may function as novel antithrombotic/anti-inflammatory agents. The detailed molecular mechanisms by which polyphosphate modulates blood clotting reactions remain to be elucidated.

Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by ␣-thrombin, ␤-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis. (Blood. 2011;118(26):6963-6970) IntroductionIn the original cascade or waterfall model of coagulation, 1 factor XI (FXI) is activated by factor XIIa (FXIIa), a member of the contact pathway of blood clotting. Patients with severe FXI deficiency may exhibit bleeding tendencies, 2,3 especially postoperative or posttraumatic bleeding in tissues with robust fibrinolytic activity. [4][5][6] Conversely, individuals with severe deficiencies in FXII, high-molecularweight kininogen, or prekallikrein do not exhibit bleeding diatheses at all, indicating that the proteins responsible for triggering the classic contact pathway of blood clotting are completely dispensable for hemostasis. 7 Thus, in normal hemostasis, FXI must be activated in vivo by a protease other than FXIIa. A solution to this conundrum was proposed in 1991 by Naito and Fujikawa 8 and by Gailani and Broze,9 who reported that thrombin up-regulates its own generation by feeding back to activate FXI, leading to a "revised model of coagulation." [9][10][11] More recently, Matafonov et al identified that ␤-thrombin and ␥-thrombin, proteolyzed derivatives of ␣-thrombin, also can activate FXI in plasma. 12 The proposal that FXI activation by thrombin plays a significant role in blood clotting in vivo is somewhat controversial. [13][14][15] In solution, the rates both of FXI activation by thrombin and of FXI autoactivation are slow but are markedly enhanced in the presence of polyanions, 8,9,16,17 although most studies have used nonphysiologic cofactors such as dextran sulfate or high concentrations of sulfatides. The relevant physiologic cofactors for FXI activation by thrombin in plasma, if any, have yet to be definitely determined.Polyphosphate (polyP), a linear polymer of inorganic phosphate residues, accumulates in a variety of microorganisms 18 and is secreted by activated human platelets. 19 We recently showed that polyP is a potent modulator of the human blood clotting system, acting at 3 points in...

Purpose: Unfractionated heparin reduces metastasis in many murine models. Multiple mechanisms are proposed, particularly anticoagulation and/or inhibition of P-selectin and L-selectin. However, the doses used are not clinically tolerable and other heparins are now commonly used. We studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. Experimental Design: Five clinically approved heparins were evaluated for inhibition of P-selectin and L-selectin binding to carcinoma cells. Pharmacokinetic studies determined optimal dosing for clinically relevant anticoagulant levels in mice. Experimental metastasis assays using carcinoma and melanoma cells investigated effects of a single injection of various heparins. Heparins were compared for structural relationships to selectin inhibition. Results: One (Tinzaparin) of three low molecular weight heparins showed increased selectin inhibitory activity, and the synthetic pentasaccharide, Fondaparinux, showed none when normalized to anticoagulant activity. Experimental metastasis models showed attenuation with unfractionated heparin and Tinzaparin, but not Fondaparinux, at clinically relevant anticoagulation levels. Tinzaparin has a small population of high molecular weight fragments not present in other low molecular weight heparins, enriched for selectin inhibitory activity. Conclusions: Heparin can attenuate metastasis at clinically relevant doses, likely by inhibiting selectins. Equivalent anticoagulation alone with Fondaparinux is ineffective. Clinically approved heparins have differing abilities to inhibit selectins, likely explained by size distribution. It should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. Caution is also raised about the current preference for smaller heparins. Despite equivalent anticoagulation, hitherto unsuspected benefits of selectin inhibition in various clinical circumstances may be unwittingly discarded.P-selectin and L-selectin are C-type lectins that recognize sialylated, fucosylated, sulfated ligands. P-selectin is stored within resting platelets and endothelial cells and translocates to the cell surface upon activation. L-selectin is constitutively expressed on most leukocyte types and mediates their interactions with endothelial ligands. Both selectins promote the initial tethering of leukocytes during extravasation at sites of inflammation. P-selectin also plays a role in hemostasis. Endogenous ligands for P-selectin and L-selectin (such as PSGL-1) are expressed on leukocytes and endothelial cells (for general reviews on selectins and their ligands, see refs. 1 -6).P-selectin and L-selectin also have pathologic roles in many diseases involving inflammation and reperfusion (7 -9), as well as in carcinoma metastasis. Many tumor cells express selectin ligands and an inverse relationship between tumor selectin ligand expression and survival has been rep...

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Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of proteinpolyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-dimetyhlaminopropyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary aminecontaining labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using 31 P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate results in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores and immobilized onto solid supports; that immobilized polyphosphate can be readily used to quantify protein binding affinities; that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting; and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues linked via phosphoanhydride bonds, is widely distributed in biology and plays important and diverse roles in nature (1,2). We recently showed that polyP is a potent modulator of the blood clotting cascade (3)(4)(5), and an expanding body of research is investigating its roles in other biological systems (6-11). Many technical obstacles remain, for investigating the biological roles of polyP and there is a real need for improved microscale methods for analyzing polyP. In particular, there is a dearth of approaches for covalently modifying polyP or attaching it to solid supports. One of the few published methods for immobilizing polyP onto surfaces is via Lewis acid/base interactions between polyP and zirconia beads (12). Although we have successfully used this method (13), it suffers from relatively high nonspecific binding of proteins to zirconia. Furthermore, this chemistry is not readily adaptable for attaching labels to polyP, or to immobilizing polyP onto the sorts of solid supports routinely used in analyses † This work was supported by grant R01 HL47014 from the National Heart, Lung and Blood institute to J.H.M., grant R01 GM75937 from the National Institute of General Medical Sciences to C.M.R., and postdoctoral fellowship grant 0920045G from the American Heart Association to R.L. Immobilization of polyP onto Polystyrene Microplate Wells and Coagulometer CuvettesA variety of reaction conditions were tested in order to optimize EDAC-mediated covalent coupling of polyP HMW to primary amines displayed on Amine Surface stripwe...

Summary Background Inorganic polyphosphates (polyP), which are secreted by activated platelets (short chain polyP) and accumulate in some bacteria (long chain polyP), support the contact activation of factor XII (FXII), and accelerate the activation of factor XI (FXI). Objectives The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation Methods and Results Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or factor IX (FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor (CTI), blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein (PK) activation, and the activation of FXI by thrombin, but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in a FIX-dependent manner. In a purified system, the activation of FXII and PK by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with CTI but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. Conclusions We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyP containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.

Summary Factor Va enhances the rate of prothrombin activation by factor Xa by four to five orders of magnitude. Production of initiating levels of factor Va from its precursor, factor V, is a critical event early in haemostasis, as factor V exhibits negligible cofactor activity. While thrombin is the most potent physiological back-activator of factor V, the first prothrombinase complexes require a source of factor Va prior to thrombin generation. A recent study by Whelihan et al. (J Thromb Haemost 2010; 8:1532–1539) identified factor XIa as a candidate for the initial thrombin-independent activation of factor V, although this reaction was slow and required relatively high concentrations of factors V and XIa. Activated platelets secrete polyphosphate, which we previously showed to be potently procoagulant. We now report that polyphosphate greatly accelerates factor V activation by factor XIa, and that this is supported by polyphosphate polymers of the size secreted by activated human platelets. This finding provides additional evidence that factor XIa-mediated generation of factor Va may contribute to the initiation of haemostasis.