Polyphosphate elicits pro‐inflammatory responses that are counteracted by activated protein C in both cellular and animal models

See also Mutch NJ. Polyphosphate scores a hat trick in regulating host defense mechanisms. This issue, pp 1142–4.

Summary.

Background:  Recent results have indicated that polyphosphate, released by activated platelets, can function as a procoagulant to modulate the proteolytic activity of serine proteases of the blood clotting cascade. Objective:  To determine whether polyphosphate is involved in inducing signal transduction in cellular and animal models. Methods:  The effect of polyphosphate on human umbilical vein endothelial cells was examined by monitoring cell permeability, apoptosis and activation of NF‐κB after treating cells with different concentrations of polyphosphate. Moreover, the expression of cell surface adhesion molecules (VCAM‐1, ICAM‐1 and E‐selectin) and the adhesion of THP‐1 cells to polyphosphate‐treated cells were monitored using established methods. In the in vivo model, the pro‐inflammatory effect of polyphosphate was assessed by monitoring vascular permeability and migration of leukocytes to the peritoneal cavity of mice injected with polyphosphate. Results:  Polyphosphate, comprised of 45, 65 and 70 phosphate units, enhanced the barrier permeability and apoptosis in cultured endothelial cells and up‐regulated the expression of cell adhesion molecules, thereby mediating the adhesion of THP‐1 cells to polyphosphate‐treated endothelial cells. These effects of polyphosphate were mediated through the activation of NF‐κB and could not be recapitulated by another anionic polymer, heparin. Polyphosphate also increased the extravasation of the bovine serum albumin (BSA)‐bound Evans blue dye and the migration of leukocytes to the mouse peritoneal cavity, which was prevented when activated protein C (APC) was intravenously (i.v.) injected 2 h before the challenge. Conclusion:  Polyphosphate, in addition to up‐regulation of coagulation, can elicit potent pro‐inflammatory responses through the activation of NF‐κB, possibly contributing to the pro‐inflammatory effect of activated platelets.     

Polyphosphate binds with high affinity to exosite II of thrombin

Summary. Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C‐terminal dodecapeptide and γ‐thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na⁺‐binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K_d approximately 5 nm ) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin‐catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin‐binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nm K_d for the polyphosphate–thrombin interaction.     

Inhibition of tissue factor pathway inhibitor by the aptamer BAX499 improves clotting of hemophilic blood and plasma

Summary. Background: Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor‐initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. Objectives: The aim of the study was to examine the concentration‐dependent augmentation of clotting by BAX499. Methods: Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet‐poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N = 55) and B (N = 11), patients with acquired hemophilia A (N = 1), and healthy controls (N = 37). Results: BAX499 significantly improved clotting of samples from hemophilic patients in a concentration‐dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. Conclusion: BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.     

Improvement of spatial fibrin formation by the anti‐TFPI aptamer BAX499: changing clot size by targeting extrinsic pathway initiation

Summary. Background: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro‐ and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high‐affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. Objective: To examine the BAX499 effect on clotting in a spatial, reaction‐diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). Methods: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. Results: BAX499 dose‐dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m^?2 by shortening lag time and increasing clot size by up to ～2‐fold. The effect was TFPI specific as confirmed by experiments in TFPI‐depleted plasma with or without TFPI supplementation. Clotting improvement was half‐maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm . The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m^?2, both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. Conclusions: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.     

Factor XIII combined with recombinant factor VIIa: a new means of treating severe hemophilia A

Summary. Background: Abnormal thrombin generation is considered the key defect in hemophilia. Conventional treatment seeks to correct this using coagulation factor replacement or bypassing agents, for example recombinant factor VIIa (rFVIIa). Previous studies demonstrate abnormal FXIII activation in patients with hemophilia. FXIII activation is essential for formation of structurally normal, stable clots. Objectives: The present study challenges the hypothesis that in hemophilia the use of plasma‐derived FXIII (pdFXIII) in combination with rFVIIa will produce a greater improvement in clot stability than promotion of thrombin generation alone. Methods: Fourteen individuals with severe hemophila A were enrolled. Whole blood was spiked ex vivo with buffer, rFVIIa (2 μg mL^?1) or rFVIIa (2 μg mL^?1) plus pdFXIII (10 μg mL^?1). Whole blood thromboelastometry assessed clot stability, after activation with tissue factor (TF) (0.15 pm ) plus tissue‐type plasminogen activator (tPa) (2 nm ). The primary outcome measure of clot stability was area under the elasticity curve (AUEC). Results: The combination of pdFXIII and rFVIIa significantly improved clot stability as measured by AUEC (P < 0.05) compared with rFVIIa alone. Conclusion: The use of pdFXIII resulted in superior clot stability compared with solely enhancing thrombin generation and we suggest that increasing thrombin generation alone fails to fully correct dysregulation of clot‐stabilizing mechanisms associated with bleeding disorders. Hemorrhage control in hemophilia may be improved using clot stabilizing drugs. FXIII shows potential as a novel agent.     

ROTEM monitoring of activated and non-activated prothrombin complex concentrate correction of dilutional coagulopathy

Objectives Prothrombin complex concentrates have been used to correct dilutional coagulopathy, but many preparations contain anticoagulants, such as heparin, to counteract their prothrombotic effects. These anticoagulants can interfere with haemostatic assays. The aim of this study was to monitor two different prothrombin complex concentrates for the treatment of albumin dilution in vitro, using rotational thromboelastometry with or without the heparin-antagonising agent protamine. Methods Citrated blood from 10 healthy volunteers was, in vitro, diluted 1:1 with 5% albumin and then corrected with a four-factor prothrombin complex concentrate with heparin anticoagulant (Confidex®) corresponding to a clinical dose of 43?IU/kg. Blood samples were tested with or without protamine. An activated prothrombin complex concentrate (APCC) (FEIBA®) without heparin in doses of 50?IU/kg and 100?IU/kg was also tested. Thromboelastometry was performed after recalcification. Results Albumin dilution significantly affected all thromboelastometry parameters. The four-factor PCC had an additional anticoagulant effect when added to the albumin-diluted blood; it was partially corrected by protamine for all parameters except maximum clot firmness. The APCC significantly improved all parameters, with over-correction of clotting time but only partial correction of maximum clot firmness. Conclusions The anticoagulant content of many prothrombin complex concentrates needs to be considered when performing in vitro testing. A heparin-free APCC better corrected an in vitro albumin-induced dilutional coagulopathy than a four-factor PCC, despite of blocking heparin with protamine.     

Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C.

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.     

Argatroban as an alternative to heparin in extracorporeal membrane oxygenation circuits

We investigated the anticoagulant effects of argatroban, a direct thrombin inhibitor, versus heparin in extracorporeal membrane oxygenation (ECMO) circuits. Three sham circuits were prepared according to our hospital's standard practice and run for six hours simultaneously. Two circuits were anticoagulated with argatroban (one with heparin in the wet prime and one without). One circuit had heparin in the initial prime and was then anticoagulated with heparin. We measured thrombin generation (prothrombin fragment 1+2, D-dimer and thrombin-antithrombin complexes), activated clotting times (ACTs) and partial thromboplastin times (aPTTs), and monitored thrombus formation using thromboelastography. ACTs were >1000 s in each circuit throughout assessment. No clot initiation was detected by thromboelastography. Thrombin generation was decreased in circuits anticoagulated with argatroban versus heparin, despite aPTTs being less prolonged. These results suggest that argatroban may be more efficacious than heparin for anticoagulation in ECMO. Additional studies are warranted to further evaluate argatroban in this setting.     

Chromogenic substrates for activated partial thromboplastin time testing: Are they worth using?

Summary In hemostasis testing the development of chromogenic substrates provides an alternative to the traditional methods based on the detection of forming clots. The new technology has often replaced the clotting tests, especially in the area of single clotting factor and inhibitor assay, less frequently for global screening tests. We report studies of the validity and clinical application of two reagents for activated partial thromboplastin time (APTT) testing with chromogenic substrates in comparison with the conventional clotting method. Congenital deficiencies of the intrinsic coagulation pathway, other than hypo- and dysfibrinogenemia detected by chromogenic APTT, agreed with those detected by the clotting APTT. The results with the two methods for plasma under heparin treatment suggest a lesser responsiveness of the chromogenic methods to heparinization. The chromogenic methods demonstrated the presence of the lupus anticoagulant in the majority of tested samples of known lupus subjects, but with a lower responsiveness than the clotting method. In conclusion, we found chromogenic APTT suitable for hemostasis testing because it generally gives the same information as the conventional clotting method with the exception of heparin monitoring and lupus anticoagulant detection, where an improved sensitivity would be desirable. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Whole blood clot formation phenotypes in hemophilia A and rare coagulation disorders. Patterns of response to recombinant factor VIIa

Summary. Until now, no routinely used clotting assay has demonstrated the power to reflect significantly a patient's response to recombinant factor (rF)VIIa. Adopting a thrombelastographic principle, profiles of continuous whole blood (WB) coagulation were studied in minimally altered WB activated with a small amount of tissue factor (TF). Investigation of the WB clotting profile was performed before and after ex vivo addition of rFVIIa 20 nm to WB from 26 patients with hemophilia A, two patients with severe hemophilia B, and individuals with deficiencies of FV, FX, FXI, and FXIII. In five patients with hemophilia plus inhibitors, the response to ex vivo added rFVIIa and to activated complex concentrate (APCC) was studied. Patients with severe and moderate hemophilia A demonstrated remarkable variance in the hemostatic characteristics at baseline, even in groups with the same FVIII:C activity levels. The response to rFVIIa at 20 nm also varied extensively, the effect correlating with the continuous WB coagulation phenotype at baseline. This indicates that the efficacy of rFVIIa may be optimized by tailoring the dose according to the hemostatic response to varying doses tested prior to in vivo administration. In patients with inhibitors against FVIII and factor IX, rFVIIa and APCC substitution resulted in quite similar response patterns that appeared to be dose dependent. In severe FV, FX, and FXIII‐deficient WB, rFVIIa addition induced minor changes only. In FXI deficiency, rFVIIa normalized the dynamic properties of clotting, although a reduced clot firmness remained unchanged. In conclusion, the thrombelastographic analysis of WB clotting, as activated with a minute amount of TF, seems an interesting method that detects phenotypic variation amongst hemophilia patients. The method appears useful for assessment of the hemostatic capacity and it seems a promising tool for evaluation of the individual response to rFVIIa or APCC before and during in vivo administration.     

Tranexamic acid combined with recombinant factor VIII increases clot resistance to accelerated fibrinolysis in severe hemophilia A

BACKGROUND: Most patients with severe hemophilia A suffer from a profoundly compromised hemostatic response. In addition to both the delayed and slow development of a clot, previous studies have documented that severe hemophilia A is also associated with reduced clot stability. OBJECTIVES: We examined whether the clot stability in hemophiliacs could be improved by treatment with tranexamic acid (TXA) in combination with recombinant factor VIII (rFVIII). PATIENTS/METHODS: Baseline blood samples were obtained from eight males with severe hemophilia A. Thereafter, a bolus injection of rFVIII was administered to increase the functional level of FVIII to approximately 50%. After 10 min, blood was collected followed by an intravenous injection of TXA. A third blood sample was obtained after a further 10 min. Whole blood clotting profiles were determined by thrombelastography using minimal tissue factor activation. Clot formation was assessed by both clot initiation and clot propagation. At clot termination, the maximum clot firmness and area under the elasticity curve were used to illustrate clot stability. Tissue-plasminogen activator was included in those experiments designed to assess clot stability. RESULTS: As expected, rFVIII increased clot formation, whereas TXA had no effect upon this parameter. Assays including tissue-plasminogen activator revealed that rFVIII increased the maximum clot firmness 3-fold; whereas the presence of TXA induced an additional 4-fold increase. The area under the elasticity curve increased 5-fold after rFVIII and 24-fold after addition of TXA. CONCLUSIONS: The study demonstrates that simultaneous treatment with TXA and rFVIII significantly improves the clot stability in patients with hemophilia A.     

Anti-Xa activity of human hepatic triglyceride lipase

Human hepatic triglyceride lipase (HTGL), purified from plasma obtained after heparin injection, markedly enhanced the anti-Xa clotting activity of normal plasma. This was shown to be caused by direct inhibition of factor Xa clotting activity by HTGL, although the amidolytic activity of factor Xa was unaffected. Preincubation of factor Xa with CaCl2 and phospholipid reduced the rate of inhibition of HTGL, indicating that phospholipid-binding sites may be involved. Heparin, and low-affinity heparin, reduced the anti-Xa activity of HTGL, suggesting that heparin and factor Xa compete for the same binding sites on the lipase molecule. These results suggest that at least part of the enhanced anti-Xa clotting activity observed after injection of heparin and heparin analogues is caused by the release of HTGL. This release could contribute toward the anticoagulant and antithrombotic actions of these drugs.     

Soluble thrombomodulin partially corrects the premature lysis defect in FVIII-deficient plasma by stimulating the activation of thrombin activatable fibrinolysis inhibitor

Summary.  Background: Previous work by others has shown that premature clot lysis occurs in plasmas deficient in components of the intrinsic pathway, due to a failure to activate thrombin activatable fibrinolysis inhibitor (TAFI). This suggests the hypothesis that bleeding in hemophilia is due not only to defective coagulation but also enhanced fibrinolysis. These studies were carried out to quantify the extent of TAFI activation over time in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP) and to determine whether soluble thrombomodulin (sTM) can correct the lysis defect in FVIII-DP. Methods: The time courses of TAFI activation in both NP and FVIII-DP were monitored after clotting with thrombin, PCPS and Ca²⁺, ± sTM. Clotting and lysis were measured turbidometrically and TAFIa using a functional assay. Results: Premature lysis that occurs in FVIII-DP is corrected by mixing deficient plasma with 10% NP. However, this does not fully correct the defect in TAFI activation. FVIII-DP must be mixed with up to 50% NP to attain the same TAFIa potential as NP. In FVIII-DP, sTM can correct the defect in TAFIa-dependent prolongation of lysis at low tPA concentrations and partially correct this defect at high tPA concentrations. Conclusions: TAFI activation increases as the concentration of FVIII increases. FVIII at a level of 10% fully corrects the lysis defect in spite of the extent of TAFI activation being only one half that obtained with 100% FVIII. In addition, sTM increases TAFI activation sufficiently to correct the premature lysis defect in FVIII-DP.     

Whole blood thrombelastographic coagulation profiles using minimal tissue factor activation can display hypercoagulation in thrombosis‐prone patients

Objective. Recently, our laboratory devised a dynamic whole blood (WB) thrombelastographic coagulation model employing activation with minute amounts of tissue factor. A series of studies were conducted to validate the feasibility of the model to illustrate hypocoagulation in various bleeding disorders and its usefulness in detecting the haemostatic effect of pro‐coagulants by ex vivo titration experiments. In this context, the present study hypothesized that the thrombelastographic model also can reveal hypercoagulation. Hence, the objective of the present study was to record dynamic WB coagulation profiles in a series of patients (n = 76) who had previously suffered from a venous (n = 34) or arterial (n = 42) thrombo‐embolic event and to compare the results with those of a group of healthy reference subjects (n = 60). Material and methods. Patients receiving vitamin K antagonist treatment were not enrolled in the study. Forty‐four of the patients had no known thrombophilia risk factor and 32 patients had at least one thrombophilia risk factor. Results. The most commonly found risk factors were mild hyperhomocysteinaemia and heterozygosity for the factor V Leiden polymorphism. The data showed that, as compared with the healthy controls, patients with a history of venous or arterial thromboembolism had a significantly greater hypercoagulant WB coagulation clot signature as defined by a shortened clotting time together with an accelerated maximum velocity of clot propagation. Conclusions. In future studies with ex vivo dose titration assessment of pro‐coagulant components mixed with blood from a patient suffering from compromised haemostasis, observation of a significantly shortened clot initiation concomitant with a distinctly accelerated clot propagation is likely to indicate an increased risk of thrombosis.     

Heparin‐induced thrombocytopenia – therapeutic concentrations of danaparoid,unlike fondaparinux and direct thrombin inhibitors,inhibit formation of platelet factor 4–heparin complexes

Summary. Background: Treatment of heparin‐induced thrombocytopenia (HIT), a disorder in which anti‐platelet factor 4 (PF4)–heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non‐heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin‐mimicking pentasaccharide). PF4–heparin complexes form at optimal stoichiometric ratios. Objectives: To compare the effects of these various non‐heparin anticoagulants in disrupting the formation of PF4–heparin complexes, and PF4‐containing immune complexes. Patients/methods: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4–heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Results: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4–heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4‐transfected HEK‐293 EBNA cell line expressing the PF4 receptor CXCR3‐B (P = 0.0408), reduced PF4–heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4–heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4–heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Conclusions: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4‐containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.     

Significance of Cross-Linking of α2-Plasmin Inhibitor to Fibrin in Inhibition of Fibrinolysis and in Hemostasis

When blood is clotted, α₂-plasmin inhibitor (α₂PI) is cross-linked to fibrin by activated fibrin-stabilizing factor (activated coagulation Factor XIII, plasma transglutaminase). The amount of cross-linked α₂-PI is proportional to the amount of α₂PI present at the time of clotting. Plasma from a patient with congenital deficiency of α₂PI was supplemented with various amounts of purified α₂PI. Clots were prepared from these plasmas and were suspended in plasma containing a normal concentration of α₂PI, and spontaneous clot lysis was observed. When the clot was formed in the presence of calcium ions and thereby allowing cross-linking to occur, the rate and extent of fibrinolysis were found to be inversely proportional to the concentrations of α₂PI present in the clot at the time of clotting. When the clot was formed in the absence of calcium ions so that no cross-linking occurred, the clot underwent fibrinolysis at similar rates, regardless of the concentrations of α₂PI in the clot. When the clot formed in the presence of calcium ions was squeezed and washed to remove unbound proteins before being suspended in plasma, the extent of fibrinolysis was also inversely proportional to the amount of α₂PI cross-linked to fibrin. Similar results were obtained when the clot was suspended in buffered saline instead of plasma. These observations suggest that spontaneous fibrinolysis is mainly carried out by plasminogen/plasminogen activator bound to fibrin, and this fibrinolysis caused by fibrin-associated activation of plasminogen was mainly inhibited by α₂PI cross-linked to fibrin. To further support this concept, α₂PI treated with activated fibrin-stabilizing factor and that had lost most of its cross-linking capacity was used in similar experiments. This modified α₂PI had the same inhibitory activity on plasmin as the native inhibitor, but gave significantly less inhibition of fibrinolysis in every experiment, particularly when the clot was compacted by platelet-mediated clot retraction or by squeezing. Thus, it was concluded that α₂PI cross-linked to fibrin plays a significant role in inhibition of physiologically occurring fibrinolysis. It is further suggested that the absence of cross-linked α₂PI contributes to accelerated fibrinolysis and hemorrhagic tendency in patients with congenital deficiency of fibrin-stabilizing factor.     

On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies.     

S35972, a direct‐acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy

Summary. Objectives: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. Methods: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. Results: S35972 inhibited human thrombin with an IC₅₀ of 3.7 nm , and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg^?1 S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg^?1 oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10 mg kg^?1 significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. Conclusions: S35972 is a non‐prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.     

Dabigatran enhances clot susceptibility to fibrinolysis by mechanisms dependent on and independent of thrombin‐activatable fibrinolysis inhibitor

Summary. Background: Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin‐activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial. Objective: To evaluate the profibrinolytic effect of dabigatran, a new, direct thrombin inhibitor, using different in vitro models. Methods and Results: The resistance of tissue factor‐induced plasma clots to fibrinolysis by exogenous tissue‐type plasminogen activator (t‐PA) (turbidimetric method) was reduced by dabigatran in a concentration‐dependent manner, with ≥ 50% shortening of lysis time at clinically relevant concentrations (1–2 μm ). A similar effect was observed in the presence of low (0.1 and 1 nm ) but not high (10 nm ) concentrations of thrombomodulin. Acceleration of clot lysis by dabigatran was associated with a reduction in TAFI activation and thrombin generation, and was largely, although not completely, negated by an inhibitor of activated TAFI, potato tuber carboxypeptidase inhibitor. The assessment of the viscoelastic properties of clots showed that those generated in the presence of dabigatran were more permeable, were less rigid, and consisted of thicker fibers. The impact of these physical changes on fibrinolysis was investigated using a model under flow conditions, which demonstrated that dabigatran made the clots markedly more susceptible to flowing t‐PA, by a mechanism that was largely TAFI‐independent. Conclusions: Dabigatran, at clinically relevant concentrations, enhances the susceptibility of plasma clots to t‐PA‐induced lysis by reducing TAFI activation and by altering the clot structure. These mechanisms might contribute to the antithrombotic activity of the drug.     

Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals.

BACKGROUND: Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. OBJECTIVES: Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. METHODS: An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. RESULTS: Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). CONCLUSIONS: Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen.