Journal of Thrombosis and Haemostasis, 6: 1750–1756 DOI: 10.1111/j.1538-7836.2008.03104.x ORIGINAL ARTICLE Polyphosphate as a general procoagulant agent S . A . S M I T H and J . H . M O R R I S S E Y Departments of Internal Medicine and Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA To cite this article: Smith SA, Morrissey JH. Polyphosphate as a general procoagulant agent. J Thromb Haemost 2008; 6: 1750–6. Summary. Background: Polyphosphate is secreted by activated platelets and we recently showed that it accelerates blood clotting, chiefly by triggering the contact pathway and promoting factor (F) V activation. Results: We now report that polyphosphate significantly shortened the clotting time of plasmas from patients with hemophilia A and B and that its procoagulant effect was additive to that of recombinant FVIIa. Polyphosphate also significantly shortened the clotting time of normal plasmas containing a variety of anticoagulant drugs, including unfractionated heparin, enoxaparin (a low molecular weight heparin), argatroban (a direct thrombin inhibitor) and rivaroxaban (a direct FXa inhibitor). Thromboelastography revealed that polyphosphate normalized the clotting dynamics of whole blood containing these anticoagulants, as indicated by changes in clot time, clot formation time, alpha angle, and maximum clot firmness. Experiments in which preformed FVa was added to plasma support the notion that polyphosphate antagonizes the anticoagulant effect of these drugs via accelerating FV activation. Polyphosphate also shortened the clotting times of plasmas from warfarin patients. Conclusion: These results suggest that polyphosphate may have utility in reversing anticoagulation and in treating bleeding episodes in patients with hemophilia. Keywords: argatroban, enoxaparin, factor V, hemophilia, heparin, rivaroxaban, warfarin. Introduction Uncontrolled hemorrhage can be a life-threatening problem for individuals with clotting factor deficiencies such as hemophilia and may also be a serious complication for patients undergoing anticoagulant therapy. Even when patients are on stable anticoagulant therapy, emergent circumstances may necessitate immediate reversal of anticoagulant status. Rapid normalization of abnormal coagulation generally requires either replacCorrespondence: Stephanie A. Smith or James H. Morrissey, College of Medicine, University of Illinois at Urbana-Champaign, 417 Med. Sci. Bldg. MC-714, 506 S. Mathews Ave., Urbana, IL 61801, USA. Tel.: +1 217 265 4036; fax: +1 217 265 5290. E-mail: sasmith6@uiuc.edu (SAS) or jhmorris@uiuc.edu (JHM) Received 16 May 2008, accepted 17 July 2008 ing missing clotting factors or administering specific antidotes [1,2]. For patients receiving warfarin, anticoagulation can be rapidly reversed via transfusing normal coagulation factors or more slowly with vitamin K therapy, while heparin can be rapidly reversed with protamine [2]. However, most newly approved anticoagulant drugs – and some under development – lack specific antidotes. Recombinant human factor (F) VIIa (rFVIIa) is approved for managing hemorrhage in patients with hemophilia with inhibitors [3,4]. More recently, in vitro studies as well as experiences with off-label use in patients suggest that rFVIIa may have general utility in reversing anticoagulant therapy [5–11], although rFVIIa administration may sometimes be associated with adverse thromboembolic events [12,13]. Currently, the primary factors limiting use of rFVIIa as a universal procoagulant are high cost and potential liability associated with off-label use. Polyphosphate (polyP) is a linear polymer of inorganic phosphate that is present in dense granules of human platelets [14,15]. PolyP is released from activated platelets and is cleared from plasma by degradation by plasma phosphatases [14,16]. We recently reported that polyP is a potent hemostatic regulator, accelerating blood coagulation by activating the contact pathway and by promoting FV activation, which in turn abrogates the anticoagulant function of tissue factor pathway inhibitor [16]. These combined effects of polyP shift the timing of thrombin generation without changing the total amount of thrombin generated. Most recently, we reported that polyP modulates fibrin clot structure, resulting in thicker fibrin fibers that are more resistant to fibrinolysis [17]. Because polyP causes an earlier burst of thrombin generation during plasma clotting, we hypothesized that polyP would also exhibit procoagulant effects under circumstances in which coagulation was impaired, including clotting factor deficiencies or anticoagulant therapy. We now report that polyP shortened the time to clot formation in normal plasma to which various anticoagulants were added in vitro. It also normalized clot dynamics in whole blood containing these anticoagulant drugs, as measured by thromboelastography. Furthermore, polyP shortened the clotting time of plasmas from patients receiving warfarin and individuals with hemophilia A or B. PolyP was as effective as adding the missing clotting factors or rFVIIa in normalizing the clotting times of hemophilia plasmas. Ó 2008 International Society on Thrombosis and Haemostasis Polyphosphate as a procoagulant 1751 Materials and methods Reagents and plasma samples Enoxaparin (Lovenox) was from Aventis (Bridgewater, NJ, USA), argatroban was from GlaxoSmithKline (Research Triangle Park, NC, USA) and rivaroxaban was a gift from Bayer HealthCare (Berkeley, CA, USA). PolyP (mean polymer size, 75 phosphate units; sold as Ôsodium phosphate glassÕ) and unfractionated heparin were from Sigma Aldrich (St Louis, MO, USA). PolyP concentrations are expressed herein in terms of phosphate monomer. Factor IX and FVa were from Enzyme Research Laboratories (South Bend, IN, USA), rFVIII (Kogenate-FSÒ) was a kind gift of Bayer HealthCare (Berkeley, CA, USA), and rFVIIa was from American Diagnostica (Stamford, CT, USA). Hemoliance RecombiplastinÒ was from Instrumentation Laboratory (Lexington, MA, USA) and InnovinÒ from Dade Behring (Newark, DE, USA). Pooled normal plasma and plasmas congenitally deficient in FVIII (<1% activity) or FIX (<1% activity) were from George King Biomedical (Overland Park, KS, USA). Factor V-immunodepleted plasma was from Haematologic Technologies (Essex Junction, VT, USA). Plasmas from patients stably anticoagulated with warfarin were from the Carle Foundation Hospital (Urbana, IL, USA). Plasmas were stored at )70 °C, thawed at 37 °C for 5 min, and then held at room temperature for no more than 30 min prior to clotting tests. Fresh whole blood for thromboelastography was collected from healthy, adult, non-smoking volunteers not receiving any medication. This study was approved by the Institutional Review Boards of the University of Illinois at Urbana-Champaign and the Carle Foundation Hospital. Written informed consent was obtained from volunteer blood donors. Plasma clotting tests Anticoagulant drugs were added to pooled normal plasma at amounts selected to span therapeutic and supratherapeutic concentrations: up to 1 U mL)1 unfractionated heparin, 100 lg mL)1 enoxaparin, 3 lg mL)1 argatroban, or 1 lg mL)1 rivaroxaban. For comparison, therapeutic ranges for unfractionated heparin and enoxaparin are generally 0.3– 0.7 and 0.6–1.0 U mL)1 (equivalent to 6–10 lg mL)1) by antiFXa activity respectively [18]. Steady-state plasma concentrations for argatroban were reported to be 0.5–0.7 lg mL)1 [19]. Mean peak plasma concentrations for rivaroxaban administered to orthopedic patients were reported to be 0.2 lg mL)1 [20]. In some experiments, clotting times of FV-deficient or pooled normal plasma spiked with FVa were evaluated with or without added anticoagulant drugs (0.4 U mL)1 unfractionated heparin, 30 lg mL)1 enoxaparin, 1 lg mL)1 argatroban, or 0.7 lg mL)1 rivaroxaban). In others, clotting times of FVIIIdeficient plasma were evaluated with or without adding up to 0.5 lg mL)1 FVIII or 20 nM FVIIa. Clotting times of FIXÓ 2008 International Society on Thrombosis and Haemostasis deficient plasmas were evaluated with or without adding up to 4 lg mL)1 FIX or 20 nM FVIIa. Plasma clotting times were quantified in 96-well polystyrene microplates (Corning Inc., Corning, NY, USA) by adding 80 lL plasma followed by 160 lL diluted thromboplastin (Recombiplastin diluted 200-fold for normal plasmas ± anticoagulant, or 8000-fold for hemophilia plasmas ± anticoagulant) in a buffer containing 100 lM sonicated liposomes (20% phosphatidylserine, 80% phosphatidylcholine), 12.5 mM CaCl2, 25 mM Tris–HCl pH 7.4, 0.1% bovine serum albumin, 150 mM NaCl. Low concentrations of tissue factor were employed to obtain significantly prolonged clotting times in the presence of anticoagulant drugs or with factor-deficient plasmas. When present, 100 lM polyP was added directly to the diluted thromboplastin. Clotting was monitored by turbidity change (A405) for 1 h at room temperature using a Spectramax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). Clotting times were calculated using SigmaPlot 7.101 (SPSS, Inc., Chicago, IL, USA) by fitting a line to the steepest segment of the absorbance curve and determining its intersection with the initial baseline A405 (representing the lag phase prior to clot formation). Assays were repeated five times. Whole blood thromboelastography Thromboelastography was performed using the ROTEMÒ four-channel system (Pentapharm, Munich, Germany) and the supplied software. Fresh, non-anticoagulated whole blood was collected via atraumatic venipuncture (discarding the initial 3 mL), then immediately transferred to the supplied disposable plastic cups (280 lL per cup) and thoroughly mixed with either 20 lL TBS (50 mM Tris–HCl pH 7.4, 150 mM NaCl) or 20 lL TBS plus the indicated additives (polyP and/or anticoagulant drugs). Each sample was divided into four cups containing: TBS only (control), polyP, anticoagulant drug, or polyP plus anticoagulant drug. Clotting was initiated by adding 20 lL diluted Innovin (in TBS) within 2 min of blood collection. Final concentrations were 87.5% whole blood, 1:17 000 dilution of Innovin, 0 or 100 lM polyP, and either no added anticoagulant drug or one of the following: 0.1 U mL)1 unfractionated heparin, 2.7 lg mL)1 enoxaparin, 1 lg mL)1 argatroban, or 0.2 lg mL)1 rivaroxaban. Measurements were continued for 2 h and thromboelastography parameters were recorded using ROTEM software. Effects of each anticoagulant were tested by adding the drug to whole blood from five different individuals. Statistical analyses Statistical analyses were performed using SigmaStat 2.03 (SPSS, Inc.). To account for inter-individual variation, thromboelastography parameters were compared using paired, twotailed t-tests with significance of P < 0.05. Pairwise comparisons for each blood donor were made between results with vs. without additive. In addition, pairwise comparisons were 1752 S. A. Smith and J. H. Morrissey performed between blood containing anticoagulant vs. blood containing anticoagulant plus polyP. (Fig. 1D), causing an approximately 80% reduction in clotting time at all rivaroxaban concentrations tested. Results PolyP reverses the anticoagulant effect of four drugs in whole blood thromboelastography Clot time (min) B Clot time (min) A 20 10 0 30 20 10 0 0.0 0.5 1.0 –1 Heparin (U mL ) in plasma 100 0 50 –1 Enoxaparin (µg mL ) in plasma Clot time (min) 10 5 Clot time (min) D 60 C 40 20 0 0 2 3 0 1 0.0 0.5 1.0 –1 Argatroban (µg mL ) in plasma Rivaroxaban (µg mL–1) in plasma Fig. 1. Polyphosphate (polyP) antagonizes the anticoagulant effect of heparin, enoxaparin, argatroban, and rivaroxaban. (A) Unfractionated heparin, (B) enoxaparin, (C) argatroban or (D) rivaroxaban were added at the indicated concentrations to pooled normal plasma, after which clotting was initiated by dilute thromboplastin. Clotting reactions contained either 100 lM polyP( ) or no polyP (s). Data are mean ± standard error (n = 5). mm A 60 40 20 0 20 40 60 2 1 4 3 2 4 1 mm B 60 40 20 0 20 40 60 C 60 40 20 0 20 40 60 D 60 40 20 0 20 40 60 0 3 2 1 4 mm We examined the ability of polyP of the size secreted by human platelets (75 phosphate units long) to reverse the anticoagulant effect of unfractionated heparin, enoxaparin (a low molecular weight heparin that acts as an indirect FXa inhibitor), argatroban (a direct thrombin inhibitor), or rivaroxaban (a direct FXa inhibitor). Drugs were added to pooled normal plasma at concentrations spanning therapeutic and supratherapeutic levels. Clotting was initiated by dilute thromboplastin, and we compared the clotting times with no added polyP to those obtained with 100 lM polyP. Each drug prolonged the clotting time in a dose-dependent manner (Fig. 1). PolyP antagonized the anticoagulant effect of both unfractionated and low molecular weight heparin, shortening the clotting time by approximately 50% at all heparin concentrations evaluated (Figs. 1A and B). This is an approximately 50% reversal of the effective heparin dose. PolyP shortened the clotting time at all argatroban concentrations tested (Fig. 1C). In the presence of polyP, concentrations of argatroban above 1 lg mL)1 failed to further prolong the clotting time. Consequently, the effects of supratherapeutic plasma levels of argatroban (1–3 lg mL)1) were blunted by polyP, resulting in a milder prolongation of clotting time equivalent to that obtained with argatroban at 0.5 lg mL)1. Of the four anticoagulant drugs tested, polyP was most effective at reversing the anticoagulant effect of rivaroxaban PolyP also reversed the anticoagulant effects of unfractionated heparin, enoxaparin, argatroban, and rivaroxaban in whole blood. As can be seen in Fig. 2A and Table 1, thromboelastography showed that adding 0.1 U mL)1 unfractionated heparin to blood prolonged both the clot time (CT) and clot formation time (CFT), and it also decreased the a angle and maximum clot firmness (MCF). PolyP shortened, but did not completely normalize CT, whereas polyP completely normalized parameters related to the kinetics of increase in clot firmness (CFT and a angle) and final clot firmness (MCF). Adding 2.7 lg mL)1 enoxaparin prolonged both CT and CFT, and it decreased a angle and MCF (Fig. 2B and Table 1). PolyP essentially normalized all four parameters. As with the heparins, adding 1 lg mL)1 argatroban prolonged both CT and CFT, and decreased the a angle and MCF (Fig. 2C and Table 1). Adding polyP normalized CFT, a angle and MFT, but CT was still prolonged. mm PolyP reverses the anticoagulant effect of four drugs in plasma clotting assays 2 4 10 1 3 3 20 30 Time (min) 40 50 Fig. 2. Whole blood thromboelastography tracings showing that polyphosphate (polyP) reverses the anticoagulant effect of heparin, enoxaparin, argatroban, and rivaroxaban. (A) Unfractionated heparin, (B) enoxaparin, (C) argatroban or (D) rivaroxaban were added to fresh whole blood. Each blood sample was divided into four aliquots receiving the following additions: (1) no additive (red curve); (2) 100 lM polyP (green curve); (3) anticoagulant (blue curve); or (4) anticoagulant plus 100 lM polyP (pink curve). Clotting was initiated with dilute thromboplastin. Ó 2008 International Society on Thrombosis and Haemostasis Polyphosphate as a procoagulant 1753 Table 1 Thromboelastography parameters Parameter No additive +Polyphosphate +Anticoagulant +Anticoagulant and polyphosphate CT (s) CFT (s) a angle (°) MCF (mm) 8.0 4.4 47.2 52.8 (0.4) (0.7) (4.3) (1.6) 5.1 1.7 69.6 63.0 (0.3)* (0.1)* (1.6)* (1.8)* Unfractionated heparin 18.8 (1.0)* 14.9 (1.7)* 18.2 (1.7)* 42.4 (2.1)* 11.5 4.5 46.8 52.4 (1.0)* (0.8) (4.6) (2.7) CT (s) CFT (s) a angle (°) MCF (mm) 6.9 3.5 53.0 57.4 (1.8) (0.6) (5.1) (4.9) 4.8 1.6 71.0 64.8 (0.6)* (0.1)* (1.2)* (2.3) * Enoxaparin 12.9 (2.2)* 7.6 (1.4)* 31.4 (5.5)* 47.0 (3.2)* 8.0 2.7 60.4 59.8 (0.7) (1.0) (7.8) (2.6) CT (s) CFT (s) a angle (°) MCF (mm) 8.1 4.4 48.0 55.0 (0.9) (1.7) (11.7) (7.3) 5.1 1.8 69.0 62.2 (0.6)* (0.5)* (6.2)* (4.8)* Argatroban 14.0 (1.3)* 6.2 (2.4)* 38.8 (9.4)* 49.4 (6.8)* 10.4 3.1 56.4 59.4 (0.9)* (0.5) (3.9) (5.3)* CT (s) CFT (s) a angle (°) MCF (mm) 7.9 3.9 49.6 55.4 (1.8) (0.8) (5.2) (7.0) 5.4 1.7 70.0 64.0 (0.5)* (0.2)* (2.4)* (4.5)* Rivaroxaban 11.7 (1.1)* 4.3 (1.3) 47.6 (8.5) 54.8 (8.6) 6.7 1.9 67.4 63.6 (0.8) (0.5)* (5.0)* (4.8)* *Significantly different from value for blood with no additive. Significantly different from value for blood containing anticoagulant without polyphosphate. CT, clot time; CFT, clot formation time; MCF, maximum clot firmness. Our previous work showed that polyP hastens thrombin generation primarily by accelerating FV activation [16]. We therefore hypothesized that polyP antagonizes the anticoagulant effects of the drugs tested above by accelerating FV activation. To test this, we examined the ability of FVa added to normal plasma to antagonize the anticoagulant effects of unfractionated heparin, enoxaparin, argatroban, or rivaroxaban. Factor Va antagonized the effects of all four drugs, with maximum reductions in clotting times equivalent to those observed with 100 lM polyP in the absence of added FVa (Fig. 3). In another test of this hypothesis, we added polyP to FV-deficient plasma that had been spiked with 1 nM FVa. This concentration of FVa will only partially abrogate the Ó 2008 International Society on Thrombosis and Haemostasis 4 2 0 8 6 4 2 0 0 4 8 PP FVa (nM) in plasma C 8 8 PP 0 4 FVa (nM) in plasma D 6 4 2 0 10 Clot time (min) Clot time (min) B 6 Clot time (min) Role of FVa A Clot time (min) Adding 0.2 lg mL)1 rivaroxaban prolonged the CT but did not significantly alter CFT, a angle or MCF (Fig. 2D and Table 1). Adding polyP plus rivaroxaban normalized the prolonged CT value. PolyP actually shifted the other thromboelastography parameters beyond those seen with native blood: it decreased the CFT in the presence of rivaroxaban to a value significantly smaller than that of native blood, and it significantly increased the a angle and MCF relative to native blood. Thus, the thromboelastography curves for blood containing polyP plus rivaroxaban were essentially identical to those for blood containing only polyP, indicating complete reversal of rivaroxaban-dependent anticoagulation. 0 4 8 PP FVa (nM) in plasma 30 20 10 0 8 PP 0 4 FVa (nM) in plasma Fig. 3. Preformed factor (F) Va is equivalent to polyphosphate (polyP) in antagonizing the anticoagulant effect of heparin, enoxaparin, argatroban, and rivaroxaban. Clotting was initiated with dilute thromboplastin using pooled normal plasma containing either the indicated anticoagulant drug (circles) or no drug (squares): (A) 0.4 U mL)1 unfractionated heparin; (B) 30 lg mL)1 enoxaparin; (C) 1 lg mL)1 argatroban; or (D) 0.7 lg mL)1 rivaroxaban. The plasmas also contained the concentrations of added FVa indicated on the x-axes (open symbols). For comparison, clotting times are presented for plasma containing 100 lM polyP but no added FVa (closed symbols, indicated as ÔPPÕ). Data are mean ± standard error (n = 5). 1754 S. A. Smith and J. H. Morrissey We evaluated the ability of polyP to reverse the prolonged clotting times of hemophilia A and B plasmas (in clotting assays using dilute thromboplastin). PolyP decreased the clotting time of plasmas from five patients with severe hemophilia A and four patients with severe hemophilia B (Figs. 5A and B). In the presence of polyP, plasmas from these patients clotted more rapidly than did normal plasma without polyP. We also evaluated the ability of polyP to reverse the prolonged clotting times of plasma from patients receiving warfarin. Adding polyP shortened, but did not completely normalize, the clotting time for all patient samples, irrespective of the patientsÕ International Normalized Ratio (Fig. 5C). We further evaluated the potential effectiveness of polyP as a procoagulant agent for hemophilia by comparing the ability of polyP to shorten the clotting times of hemophilia A or B plasmas to which the missing clotting factor was replenished, or to which rFVIIa was added. Adding 100 lM polyP shortened the plasma clotting times in both FVIII deficiency and FIX deficiency to a greater degree than did replacement of the missing clotting factor (Figs. 6A and C). PolyP also shortened the plasma clotting time to an extent similar to that observed Clot time (min) 30 20 10 0 N U E A R Fig. 4. In the absence of factor (F) V, polyphosphate (polyP) no longer antagonizes the anticoagulant effect of heparin, enoxaparin, argatroban or rivaroxaban. Anticoagulants were added to FV-deficient plasma containing 1 nM FVa, after which clotting was initiated by dilute thromboplastin. Anticoagulants were: (U) 0.4 U mL)1 unfractionated heparin; (E) 30 lg mL)1 enoxaparin; (A) 2 lg mL)1 argatroban; (R) 0.7 lg mL)1 rivaroxaban; or (N) no additive. Wells without added polyP (open bars) were compared to those containing 100 lM polyP (filled bars). Data are mean ± standard error (n = 5). Clot time (min) 20 10 0 P1 P2 P3 P4 P5 Hemophilia A patients N P7 P8 P9 Hemophilia B patients N B Clot time (min) PolyP shortens the clotting times of hemophilia plasmas and plasmas from warfarin patients A 30 40 20 0 P6 C Clot time (min) anticoagulant function of the tested drugs (see Fig. 3). Furthermore, as this plasma contains no FV, polyP will be unable to promote any further FVa generation; we therefore hypothesized that polyP should be without effect on clotting times. Indeed, we found that adding 100 lM polyP to such plasma mixtures failed to antagonize the anticoagulant effects of any of the four drugs tested (Fig. 4). These results argue that the polyP antagonizes the anticoagulant function of these four drugs solely by its ability to promote FV activation. 10 8 6 4 2 0 N 1.7 2.3 2.8 3.2 3.7 4.3 4.8 Patients receiving warfarin (INR value) Fig. 5. Polyphosphate (polyP) shortens the clotting time of hemophilia plasmas and plasmas from patients receiving warfarin. Clotting tests were performed with plasmas from (A) five different patients with hemophilia A; (B) four different patients with hemophilia B; or (C) seven different patients receiving warfarin therapy (with the indicated International Normalized Ratio values). ÔNÕ indicates data from pooled normal plasma. Clotting reactions without added polyP (open bars) were compared to those containing 100 lM polyP (filled bars). Data are mean ± standard error (n = 5). when rFVIIa was added to the plasmas (Figs. 6B and D). Thus, in this clotting assay, the effectiveness of 100 lM polyP was comparable to that of 6 nM rFVIIa in FIX-deficient plasma or 15 nM rFVIIa in FVIII-deficient plasma. We also examined the combination of adding polyP along with FIX, FVIII or rFVIIa and we found that effects of 100 lM polyP were additive to those of rFVIIa (Fig. 6). Discussion We have now demonstrated that polyP of the size secreted by human platelets partially or completely reversed the anticoagulant effects of four drugs (unfractionated heparin, enoxaparin, argatroban, and rivaroxaban). PolyP did this in spite of the differing modes of action of these drugs, and was particularly Ó 2008 International Society on Thrombosis and Haemostasis A 30 B Clot time (min) Clot time (min) Polyphosphate as a procoagulant 1755 20 10 0 0.0 0.1 0.2 0.3 0.4 0.5 FVIII (µg mL–1) added to FVIII deficient plasma 20 10 0 ne fM pM pM pM nM nM No 200 2 20 200 2 20 FVIIa added to FVIII deficient plasma D Clot time (min) Clot time (min) C 30 40 20 0 40 20 0 1 0 2 3 4 FIX (µg mL–1) added to FIX deficient plasma ne fM pM pM pM nM nM No 200 2 20 200 2 20 FVIIa added to FIX deficient plasma Fig. 6. Ability of polyphosphate (polyP) to shorten the clotting time of plasma from patients with hemophilia compared with the effect of supplementing the plasmas with the missing clotting factor or recombinant factor (F) VIIa (rFVIIa). (A) Factor VIII or (B) rFVIIa was added to plasma from a patient with hemophilia A. (C) Factor IX or (D) rFVIIa was added to plasma from a patient with hemophilia B. Clotting was initiated by dilute thromboplastin. Clotting reactions without added polyP (s) were compared to those containing 100 lM polyP ( ). Data are mean ± standard error (n = 5). Factor VIII:1IU mL)1 = 0.25 lg mL)1. Factor IX: 1 IU mL)1 = 5.13 lg mL)1. For comparison, the mean clotting time for pooled normal plasma was 12.2 min. effective at reversing anticoagulation because of the direct FXa inhibitor, rivaroxaban. Thromboelastography confirmed that polyP shortened the time to initial clot formation (CT) in whole blood. The anticoagulant effects of unfractionated heparin, enoxaparin, or argatroban were not entirely reversed by polyP, whereas adding polyP to blood containing rivaroxaban resulted in a procoagulant effect similar to that observed in the absence of the anticoagulant drug. Because the majority of thrombin is generated after initial clot formation, thromboelastography provided additional information regarding the dynamics of clot formation as it occurred after initial clot detection in plasma clotting assays. Despite the lack of normalization of CT by polyP in blood anticoagulated with heparin, enoxaparin, and argatroban, polyP completely reversed the effects of these anticoagulants on the parameters associated with the dynamics of clot formation that indicate propagation and rigidity (CFT, a angle, and MCF). Interestingly, when these anticoagulant drugs were added to plasma in which all the FV was already in the active state (FVa), polyP failed to reverse the anticoagulant effects of any of the drugs. Furthermore, simply adding FVa to normal plasma antagonized the anticoagulant effect of these drugs to a level similar to that observed with polyP. Taken together, these results argue that polyP abrogates anticoagulant drug activity by enhancing FVa generation [16]. The ability of polyP to antagonize argatroban was of particular interest, as a direct Ó 2008 International Society on Thrombosis and Haemostasis thrombin inhibitor should act only downstream of the prothrombinase complex. However, an important function of thrombin is the feedback activation of FV to FVa, a process that should be slowed by argatroban (thus delaying clotting), but we previously showed that polyP accelerates the rate of FV activation by both FXa and thrombin [16]. We therefore propose that polyP works to reverse argatrobanÕs anticoagulant function by accelerating FV activation. We also found that polyP shortened the clotting of hemophilic plasmas or plasmas from patients receiving warfarin, although it only partially normalized the clotting times of the latter group. PolyP may have incompletely reversed the effects of vitamin K antagonists because enhanced FVa generation was unable to fully augment the assembly of the prothrombinase complex when undercarboxylated coagulation factors were present. In contrast, tissue factor-triggered coagulation was completely normalized by polyP when either FVIII or FIX were deficient, and in fact polyP was as effective at normalizing the clotting time of hemophilic plasmas as was replacing the missing clotting factor or adding rFVIIa, both of which are accepted in vivo therapies for managing hemorrhage in patients with hemophilia. It is of particular interest that the procoagulant effect of polyP was additive to that of rFVIIa. Because polyP acts at a later level in coagulation than does FVIIa, the two treatments may be complementary. As the costs associated with treating bleeding episodes in hemophilia with rFVIIa are so large, even a minor reduction in rFVIIa dose could profoundly reduce the cost of managing these patients. Recently, rFVIIa has been proposed as a Ôuniversal procoagulantÕ with the potential to reverse the anticoagulant activity of a variety of drugs [1,3,21]. In studies in vitro or ex vivo, rFVIIa partially or completely reversed the anticoagulant effects of unfractionated heparin [9] enoxaparin [9], fondaparinux [9,10], warfarin [22], argatroban [9], or bivalirudin [9]. In vivo data regarding reversal of anticoagulation with rFVIIa are limited, although rFVIIa antagonized the anticoagulant effects of fondaparinux [11,23] and idraparinux [24] in normal healthy volunteers. In anecdotal reports and case series, rFVIIa normalized in vitro clotting and improved or prevented bleeding in patients receiving unfractionated heparin [25], low molecular weight heparin [5,25], vitamin K antagonists [7,8,25– 29] or lepirudin [6]. Despite frequent anecdotal reports of using rFVIIa to reverse anticoagulant-induced hemorrhage, the drug is not currently approved for this indication. Controlled studies evaluating its safety and effectiveness in comparison to other therapies are lacking, and appropriate dose protocols have yet to be clearly determined [3]. Furthermore, thromboembolic adverse events following administration of rFVIIa have been described [12], the majority of which are associated with offlabel use. Lastly, rFVIIa is expensive. PolyP added to plasma has a half-life of approximately 1.5 h, apparently being degraded by plasma phosphatases [16]. Few data are available for polyP metabolism and clearance in vivo, although 99mTc-labeled polyP was reported to distribute to bone [30]. Therapeutic use of polyP for its effects on coagulation may possibly necessitate continuous or multiple 1756 S. A. Smith and J. H. Morrissey infusions, but polyP is inexpensive and may therefore represent a significant cost savings over other available therapies. The present studies demonstrate that polyP partially or completely reversed the effects of a variety of anticoagulant drugs in vitro, and shortened the clotting time of plasmas deficient in FVIII or FIX. These findings suggest that polyP might be useful for treatment of bleeding episodes in patients with compromised hemostatic systems, although in vivo tests would clearly be needed to establish this. 12 13 14 15 Acknowledgements We thank Bayer for providing Kogenate and Rivaroxaban, and Diagnostica Stago-US for generously loaning the ROTEM system. 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