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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