Novel insights into the regulation of coagulation by factor V isoforms,tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro‐ and anticoagulant pathways. It circulates as a single‐chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane‐bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV‐Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV‐Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C‐terminus of the FV‐Short B domain, which binds the positively charged C‐terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV‐Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.     

Comparison of the anticoagulant intensities of fondaparinux and enoxaparin in the organization to assess strategies in acute ischemic syndromes (OASIS)‐5 trial

Summary. Background: In the OASIS‐5 trial, fondaparinux reduced major bleeding with similar short‐term efficacy as enoxaparin but lowered death and stroke during long‐term follow‐up. The mechanism of lower bleeding and improved efficacy with fondaparinux is uncertain. Methods and Results: We compared the anti‐Xa concentration (reflecting drug levels), Xa clot time (reflecting anticoagulant effect) and endogenous thrombin potential (ETP; a global test of hemostatic function) in plasma samples collected 6, 24 and 72 h after the first dose of the study drug in 48 patients randomly assigned fondaparinux 2.5 mg day^?1 and 42 patients assigned enoxaparin 1 mg kg^?1 twice daily in the OASIS‐5 trial. Patients assigned to fondaparinux compared with enoxaparin had a significantly lower mean anti‐Xa level [0.52 IU mL^?1 (SD 0.22 IU mL^?1) vs. 1.2 IU mL^?1 (SD 0.45 IU mL^?1), P < 0.0001] and Xa clot time [64.9 s (SD 17.7 s) vs. 111.8 s (SD 29.6 s), P < 0.0001], and significantly higher ETP area under the curve (AUC) [386.7 mA (SD 51.5 mA) vs. 206.4 mA (SD 90.6 mA), P < 0.001] at 6 h, and these differences remained evident at 24 and 72 h. There was significantly less variability of the results of anti‐Xa levels, Xa clot time and ETP AUC for fondaparinux compared with enoxaparin at 6 h (P < 0.001 for each comparison). Conclusion: Fondaparinux 2.5 mg day^?1 compared with enoxaparin 1 mg kg^?1 twice daily produces less variable anticoagulant effect and lower mean anticoagulant intensity. These results most likely explain the reduced risk of bleeding seen with fondaparinux compared with enoxaparin in the OASIS‐5 trial and suggest that a lower intensity of anticoagulation than used in the past may be sufficient to prevent recurrent ischemic events and death in patients with ACS who are concurrently treated with aspirin and clopidogrel.     

Factor Xa or thrombin: is factor Xa a better target?

Summary.  Existing vitamin K antagonists (VKAs) have drawbacks that limit their effectiveness, safety, and overall frequency of use. Oral anticoagulants in development with targeted action against individual coagulation factors, specifically direct factor (F) Xa and IIa inhibitors, appear to have pharmacokinetic and pharmacodynamic properties that overcome the limitations of the VKAs. Based on the theory of how coagulation factors interact, on the results of in vitro studies, and on clinical outcomes, there is accumulating evidence that FXa may represent a better target for inhibition than FIIa. This is based on an understanding of the amplified nature of coagulation factor interactions and fibrin formation, the need for smaller doses of an anticoagulant to block coagulation progression earlier in the sequence of reactions, the evidence for incomplete suppression of thrombin generation with direct thrombin inhibitors, evidence for rebound hypercoagulability with thrombin inhibitors, and clinical results with the indirect, parenteral, FXa inhibitor (fondaparinux), as well as early phase II results of new oral Xa and IIa inhibitors compared with enoxaparin. The latter studies, although not comparative, provide some evidence for the effectiveness and safety of Xa inhibitors at a range of doses not seen with the direct IIa inhibitors.     

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin‐independent mechanism

Summary. Background: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin‐independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity. Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC₅₀s) of low molecular weight heparin (LMWH), super‐sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX‐dependent and FIX‐independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock‐depleted or antithrombin/FIX‐depleted plasma supplemented with recombinant FIX. Results: UFH and fondaparinux demonstrated similar potency under FIX‐dependent and FIX‐independent conditions, whereas LMWH (2.9‐fold) and ssLMWH (5.1‐fold) demonstrated increased potency with limiting TF. UFH (62‐fold) and fondaparinux (42‐fold) demonstrated markedly increased EC₅₀ values in antithrombin‐depleted plasma, whereas LMWH (9.4‐fold) and ssLMWH (two‐fold) were less affected, with an EC₅₀ within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin‐depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin‐binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin‐independent interaction with the FIXa heparin‐binding exosite.     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy

Summary. Objective: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long‐lasting antithrombin (AT)‐dependent anti‐factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. Methods: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. Results: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K_d < 1 nm ) and showed a potent anti‐FXa effect and inhibition of thrombin generation with IC₅₀ values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long‐lasting anti‐FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. Conclusions: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former’s pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.     

Antithrombotic properties of a direct thrombin inhibitor with a prolonged half-life and AT-mediated factor Xa inhibitory activity

Summary.  Rebound thrombin generation after successful thrombolysis might be related to (i) too short-term anticoagulant therapy and to (ii) the inability of heparin derivatives to inhibit clot-bound thrombin. To meet these shortcomings, a compound was synthesized, which consists of a pentasaccharide conjugated to a direct thrombin inhibitor. This compound (Org 42675) has a 10 times longer half-life compared with the original half-life of the direct thrombin inhibitor, while the thrombin inhibitory activity is maintained. An extra advantage of this product is the inhibitory activity on thrombin generation via antithrombin III (AT)-mediated factor (F)Xa inhibition. Org 42675 inhibited in vitro clot-bound thrombin with similar activity to the direct thrombin inhibitor argatroban. In experimental models in rats, Org 42675 showed on a molar base similar antithrombotic activity to unfractionated heparin, was more active than argatroban and was more active than fondaparinux sodium (AT-mediated FXa inhibitor) in arterial thrombosis. Finally, Org 42675 was far more active than the three reference compounds in an experimental thrombolysis model in rabbits. These properties of Org 42675, with its FXa and (clot-bound) thrombin inhibitory activity in combination with its long half-life, make this compound a powerful drug that is likely to be effective in the prevention of re-occlusion after successful thrombolysis in man.     

Zn2+‐containing protein S inhibits extrinsic factor X‐activating complex independently of tissue factor pathway inhibitor

Summary. Background: Protein S (PS) has direct anticoagulant activity, independently of activated protein C (APC). The mechanisms underlying this activity remain unclear, because PS preparations differ in activity, giving rise to conflicting results. Some purification procedures result in loss of intramolecular Zn²⁺, which is essential for inhibition of prothrombinase. Objective: To investigate the inhibition of extrinsic factor (F)Xase by Zn²⁺‐containing PS. Methods: Purified component extrinsic FXase assays were used to determine FXa generation in the presence and absence of PS and/or tissue factor pathway inhibitor (TFPI). Binding assays, immunoblots and thrombin generation assays in plasma supported the FXase data. Results: Zn²⁺‐containing PS potently inhibited extrinsic FXase in the presence of saturating phospholipids, independently of TFPI, whereas inhibition of extrinsic FXase by Zn²⁺‐deficient PS required TFPI. Immunoblots for FXa and functional assays showed that Zn²⁺‐containing PS inhibited primarily the quantity of FXa formed by tissue factor (TF)–FVIIa, rather than FXa amidolytic activity. Zn²⁺‐containing PS, but not Zn²⁺‐deficient PS, bound to TF with high affinity (K_{d app} = 41 nm ) and targeted TF function. Binding of PS to FVIIa was negligible, whereas PS showed appreciable binding to FX. Increasing FX concentrations 10‐fold reduced PS inhibition five‐fold, suggesting that PS inhibition of FXase is FX‐dependent. PS also exhibited TFPI‐independent and APC‐independent anticoagulant activity during TF‐initiated thrombin generation in plasma. Conclusions: PS that retains native Zn²⁺ also retains anticoagulant functions independently of APC and TFPI. Inhibition of extrinsic FXase by PS at saturating levels of phospholipids depends on PS retention of intramolecular Zn²⁺, interaction with FX, and, particularly, interaction with TF.     

Effect of the anti-factor Xa and anti-factor IIa activities of low-molecular-weight heparins upon the phases of thrombin generation

BACKGROUND: Low-molecular-weight heparins (LMWHs), derived from unfractionated heparin (UFH) by different depolymerization procedures, vary in both their relative abilities to enhance the inhibition of FXa (anti-FXa) and thrombin (anti-FIIa), and in their physicochemical properties. OBJECTIVE: We aimed to profile the inhibition of thrombin generation induced by bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin in platelet-rich plasma (PRP), and to compare them with UFH and fondaparinux (a synthetic pentasaccharide that specifically enhances FXa inhibition). METHODS: Different LMWHs, UFH or fondaparinux were added to normal PRP. Thereafter, tissue factor-triggered thrombin generation was assessed using the Thrombogram-Thrombinoscope assay. RESULTS: At equivalent anti-FIIa activity concentrations, LMWHs and UFH exhibited similar inhibitory effects upon thrombin generation. However, when used at equivalent anti-FXa activity concentrations, tinzaparin was significantly more active than the other LMWHs at inhibiting thrombin generation, and had similar activity to that of UFH. Enoxaparin, nadroparin and dalteparin all showed similar inhibitory activities. In these experiments, bemiparin exhibited the lowest inhibitory effect on thrombin generation of all the LMWHs. At 0.1 microg mL(-1) (0.093 anti-FXa IU mL(-1)), fondaparinux inhibited the rate of thrombin generation by 50%. A 7-fold higher concentration of fondaparinux was required to inhibit the endogenous thrombin potential by 50%. CONCLUSIONS: LMWHs have a variable inhibitory effect on thrombin generation in vitro when compared by anti-FXa activity, but are similar when compared by their anti-FIIa activities. The rate of thrombin generation during the propagation phase, rather than the endogenous thrombin potential, is more sensitive to the anticoagulant activity of fondaparinux and the polysaccharide chains of LMWHs possessing only anti-FXa activity.     

Proteolytic cleavage of protein S during the hemostatic response

BACKGROUND: Protein S is a vitamin K-dependent protein with anticoagulant properties. It contains a so-called thrombin-sensitive region (TSR), which is susceptible to cleavage by coagulation factor Xa (FXa) and thrombin. Upon cleavage, the anticoagulant activity of protein S is abolished. OBJECTIVE: The aim of the present study was to determine whether protein S is cleaved within the TSR during activation of the coagulation system under near physiological conditions. RESULTS: In a reconstituted coagulation system containing apart from protein S only procoagulant constituents and synthetic phospholipid vesicles, protein S was cleaved at Arg60 by the FXa generated (3 mol min(-1) mol(-1) enzyme). FXa-catalyzed cleavage of protein S, however, was inhibited by factor Va and prothrombin by more than 70%. During clotting of recalcified citrated plasma in the presence of a synthetic lipid membrane, no FXa-catalyzed proteolysis of protein S was observed. Substituting platelets for phospholipid vesicles resulted both in the reconstituted system and in plasma in cleavage of the TSR. Cleavage was at Arg60 and was observed upon platelet activation, irrespective of the presence of FXa (13 pmol min(-1) 10(-8) platelets). No cleavage by thrombin was observed in either the reconstituted coagulation system or clotting plasma. CONCLUSION: These findings suggest that in vivo the anticoagulant activity of protein S is not down-regulated by FXa or thrombin during activation of coagulation. Our results rather suggest a role for a platelet protease in down-regulating the anticoagulant activity of protein S during the hemostatic response.     

A randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor IXa inhibitor

Summary. Background: Active and safe reversibility of anticoagulation is an unmet need in clinical care. Factor IXa, required for rapid thrombin generation on platelet surfaces, is a novel target for modulating coagulation. REG1 comprises RB006 (drug) and RB007 (antidote). RB006, a ribonucleic acid aptamer, exerts its anticoagulant effect by selectively binding FIXa. RB007, the complementary oligonucleotide antidote, binds to RB006 by Watson–Crick base pairing, neutralizing its anti‐FIXa activity. Objective: To test the multiple repeat‐dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of REG1. Methods: We randomized 39 healthy volunteers to receive either three consecutive weight‐adjusted, drug–antidote treatment cycles, or double placebo. Each treatment cycle included an intravenous bolus of 0.75 mg kg^?1 RB006, followed 60 min later by a descending dose of RB007, ranging from a 2 : 1 to 0.125 : 1 antidote/drug ratio (1.5 mg kg^?1 to 0.094 mg kg^?1 RB007). Serial clinical assessments and coagulation measurements were performed through 14 days postrandomization. Results: Repeat doses of RB006 achieved highly reproducible activated partial thromboplastin time (APTT) levels with low intrasubject variability (coefficient of variation 5.5%, intraclass correlation coefficient 5.8 at 15 min postdose), while repeat doses of RB007 reversed the APTT levels dose‐dependently and reproducibly. There was no major bleeding and there were no other serious adverse events. Conclusions: This is the first human study demonstrating multiple repeat‐dose safety, intraindividual pharmacodynamic reproducibility and graded active reversibility of an RNA aptamer–oligonucleotide antidote pair. The results lay the foundation for studying the translation of this novel anticoagulation platform to a wide variety of clinical applications.     

A high affinity,antidote‐controllable prothrombin and thrombin‐binding RNA aptamer inhibits thrombin generation and thrombin activity

Background:  The conversion of prothrombin to thrombin is one of two non‐duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti‐thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D‐14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. Objectives: We sought to explore the structure of R9D‐14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA_R9D‐14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results:  RNA_R9D‐14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I‐mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA_R9D‐14T significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC‐183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA_R9D‐14T anticoagulation in vitro. Conclusions:  Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA_R9D‐14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.     

Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies

Summary. Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late‐stage clinical development for the prevention and treatment of thromboembolic diseases. Objective: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. Methods: Studies were conducted in arteriovenous‐shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. Results: In vitro, apixaban is potent and selective, with a K_i of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K_i (nm ): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC_2× (μm , concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 μm did not alter human and rabbit platelet aggregation to ADP, γ‐thrombin, and collagen. In vivo, the values for antithrombotic ED₅₀ (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED_3× (dose that increased BT by 3‐fold) were 0.27 ± 0.03, 0.11 ± 0.03, 0.07 ± 0.02 and > 3 mg kg^?1 h^?1 i.v. for apixaban, 0.05 ± 0.01, 0.05 ± 0.01, 0.27 ± 0.08 and > 3 mg kg^?1 h^?1 i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 ± 0.04, 0.27 ± 0.01, 0.08 ± 0.01 and 0.70 ± 0.07 mg kg^?1 day^?1 p.o. for the oral anticoagulant warfarin, respectively. Conclusions: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.     

Fondaparinux treatment of acute heparin‐induced thrombocytopenia confirmed by the serotonin‐release assay: a 30‐month, 16‐patient case series

See also Greinacher A. Immunogenic but effective: the HIT‐fondaparinux brain puzzler. This issue, pp 2386–8; Goldfarb MJ, Blostein MD. Fondaparinux in acute heparin‐induced thrombocytopenia: a case series. This issue, pp 2501–3. Summary. Background: Fondaparinux is theoretically an attractive agent for the treatment of immune heparin‐induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet‐activating anti‐platelet factor 4/heparin antibodies. Although reports of the use of fondaparinux for this indication have thus far been favorable, the diagnosis of HIT in most cases was not based on definitive laboratory confirmation of heparin‐dependent, platelet‐activating antibodies. Objectives: To report thrombotic and major bleeding outcomes with fondaparinux in patients with a high likelihood of having acute HIT based on clinical features and a positive result in the confirmatory platelet serotonin‐release assay (SRA), a sensitive and specific test for platelet‐activating HIT antibodies. Methods/Patients: We reviewed consecutive eligible patients with SRA‐positive HIT (mean peak serotonin release, 91% [normal, < 20%]; mean IgG‐specific PF4/heparin enzyme immunoassay result, 2.53 optical density units [normal, < 0.45 units]) in one medical center over a 30‐month period who received fondaparinux for anticoagulation during acute HIT (platelet count, < 150 × 10⁹ L^?1). Where available, plasma samples were used to measure thrombin–antithrombin (TAT) complex levels. Results: Sixteen patients with SRA‐positive HIT received fondaparinux: 14 surgical (11 after cardiac surgery; three after vascular surgery) and two medical (acute stroke). Fifty‐six per cent of patients had HIT‐associated thrombosis at the time of diagnosis. No patient developed new, recurrent or progressive thrombosis; one patient developed a major bleed (calf hematoma). One patient judged to have irreversible tissue necrosis before receiving fondaparinux therapy ultimately required limb amputation. TAT complex levels were reduced within 24 h of starting fondaparinux, and 13 of 13 patients were successfully switched to warfarin. Conclusion: Fondaparinux shows promise for the treatment of patients with SRA‐positive acute HIT.     

Coagulation procofactor activation by factor XIa

Summary. Background: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca²⁺. FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by ～ 10⁷. One potential APTT procofactor activator in this setting is FXIa. Objective: To test the hypothesis that FXIa can activate FVIII and FV. Methods: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Results: Kinetic analyses of FXIa‐catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. Conclusions: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.     

Thrombin‐inhibiting perfluorocarbon nanoparticles provide a novel strategy for the treatment and magnetic resonance imaging of acute thrombosis

Summary. Background: As a regulator of the penultimate step in the coagulation cascade, thrombin represents a principal target of direct and specific anticoagulants. Objective: A potent thrombin inhibitor complexed with a colloidal nanoparticle was devised as a first‐in‐class anticoagulant with prolonged and highly localized therapeutic impact conferred by its multivalent thrombin‐absorbing particle surface. Methods: PPACK (Phe[D]‐Pro‐Arg‐Chloromethylketone) was secured covalently to the surface of perfluorocarbon‐core nanoparticle structures. PPACK and PPACK nanoparticle inhibition of thrombin were assessed in vitro via thrombin activity against a chromogenic substrate. In vivo antithrombotic activity of PPACK, heparin, non‐functionalized nanoparticles and PPACK nanoparticles was assessed through intravenous (i.v.) administration prior to acute photochemical injury of the common carotid artery. Perfluorocarbon particle retention in extracted carotid arteries from injured mice was assessed via ¹⁹F magnetic resonance spectroscopy (MRS) and imaging (MRI) at 11.7 T. Activated partial thromboplastin time (APTT) measurements determined the systemic effects of the PPACK nanoparticles at various times after injection. Results: An optical assay verified that PPACK nanoparticles exceeded PPACK’s intrinsic activity against thrombin. Application of an in vivo acute arterial thrombosis model demonstrated that PPACK nanoparticles outperformed both heparin (P = 0.001) and uncomplexed PPACK (P = 0.0006) in inhibiting thrombosis. ¹⁹F MRS confirmed that PPACK nanoparticles specifically bound to sites of acute thrombotic injury. APTT normalized within 20 min of PPACK nanoparticles injection. Conclusions: PPACK nanoparticles present thrombin‐inhibiting surfaces at sites of acutely forming thrombi that continue to manifest local clot inhibition even as systemic effects rapidly diminish and thus represent a new platform for localized control of acute thrombosis.     

Anti‐PF4/heparin antibody formation postorthopedic surgery thromboprophylaxis: the role of non‐drug risk factors and evidence for a stoichiometry‐based model of immunization

Summary. Background: Heparin‐induced thrombocytopenia is an antibody‐mediated disorder exhibiting variable frequency in different clinical settings. Antibodies recognize PF4/heparin complexes formed at optimal stoichiometric molar ratios. Objective: To identify clinical factors influencing risk of anti‐PF4/heparin immunization. Patients/methods: We performed observational studies and exploratory analyses of the frequency of anti‐PF4/heparin antibody formation in 6324 patients who received enoxaparin or fondaparinux in four randomized controlled trials of postorthopedic surgery thromboprophylaxis. Variables included surgery type (knee vs. hip), timing of first anticoagulant dose (pre‐ vs. postsurgery), circumstances of surgery (elective vs. hip fracture), anticoagulant (enoxaparin vs. fondaparinux) and body‐mass index (BMI). We applied a stoichiometry‐based model that predicts immunization risk based on expected differences in PF4/anticoagulant ratios in different settings, and specifically used this model to predict the effect of increasing BMI quartiles upon relative risk (RR) of immunization for fondaparinux vs. enoxaparin. Results: Anti‐PF4/heparin immunization was more frequent after knee vs. hip surgery (particularly for enoxaparin), and when enoxaparin was given post‐ rather than pre‐elective surgery; however, the opposite occurred with hip fracture surgery, that is, antibody formation was more frequent when enoxaparin or fondaparinux was given presurgery. The RR of immunization for fondaparinux vs. enoxaparin decreased significantly for increasing BMI quartiles, an effect predominantly because of increasing immunization with enoxaparin at increasing BMI quartiles. Conclusions: Several non‐drug factors – including type and circumstances of surgery, timing of first anticoagulant dose and BMI – influence risk of anti‐PF4/heparin antibody formation, consistent with a stoichiometry‐based immunization model of PF4 and anticoagulant ratios occurring during the early peri‐operative period.     

Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition.

BACKGROUND: Acute coronary syndrome (ACS) is associated with thrombin formation, triggered by ruptured or eroded coronary atheroma. We investigated whether thrombin generation based on circulating coagulation protein levels, could distinguish between acute and stable coronary artery disease (CAD). METHODS AND RESULTS: Plasma coagulation factor (F) compositions from 28 patients with ACS were obtained after onset of chest pain. Similar data were obtained from 25 age- and sex-matched patients with stable CAD. All individuals took aspirin. Patients on anticoagulant therapy were excluded. The groups were similar in demographic characteristics, comorbidities and concomitant treatment. Using each individual's coagulation protein composition, tissue factor (TF) initiated thrombin generation was assessed both computationally and empirically. TF pathway inhibitor (TFPI), antithrombin (AT), factor II (FII) and FVIII differed significantly (P < 0.01) between the groups, with levels of FII, FVIII and TFPI higher and AT lower in ACS patients. When thrombin generation profiles from individuals in each group were compared, simulated maximum thrombin levels (P < 0.01) and rates (P < 0.01) were 50% higher with ACS while the initiation phases of thrombin generation were shorter. Empirical reconstructions of the populations reproduced the thrombin generation profiles generated by the computational model. The differences between the thrombin generation profiles for each population were primarily dependent upon the collective contribution of AT, FII and FVIII. CONCLUSION: Simulations of thrombin formation based on plasma composition can discriminate between acute and stable CAD.     

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Summary.  Background:  The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives:  We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results:  TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion:  Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.     

The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.