The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX

Summary.  Patients with hemophilia have an impaired thrombin generation and therefore form loose fibrin hemostatic plugs that are easily dissolved by fibrinolysis. This prevents maintained hemostasis in these patients, resulting in a severe bleeding disorder. Recombinant (F)VIIa has been shown to enhance thrombin generation on already thrombin-activated platelets in the absence of FVIII and FIX. An efficacy rate of 80–90% has been found in hemophilia patients with inhibitors against FVIII or FIX both in association with major surgery and in the treatment of serious bleedings. In a model measuring fibrin clot permeability in a platelet-containing system described by Blombäck et al . (1994) this was demonstrated to be dependent on the concentration of FVIII and FIX. The addition of rFVIIa in concentrations of 1.9, 4.8 and 9.6 µg mL⁻¹ normalized fibrin clot permeability. The concentration of 1.9 µg mL⁻¹ of rFVIIa normalized clot permeability in this system and the higher concentrations of rFVIIa added only slightly to the effect. No further decrease in clot permeability was found when rFVIIa in a concentration of 1.9 µg mL⁻¹ was added to a sample with a normal concentration (100%) of FVIII or FIX. Higher concentrations of rFVIIa added to the plasma containing 100% of FVIII or FIX induced only a slight further decrease of fibrin permeability constant, arguing against any unwanted effect of extra rFVIIa on clot permeability in the case of a normal hemostasis. Furthermore, the fibrin network was studied with 3D microscopy and the loose network found in the absence of FVIII or FIX increased in density with increasing FVIII or FIX concentrations. The addition of rFVIIa to FVIII- or FIX-deficient systems altered the network structure, making the fibers thinner and more tightly packed.     

Sulfatides inhibit platelet adhesion to von Willebrand factor in flowing blood

Summary.  Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins such as von Willebrand factor (VWF), P-selectin, laminin and thrombospondin. Previous studies have localized the sulfatide-binding site of VWF to amino acid residues Gln626–Val646 in the A1 domain. The A1 domain also contains the binding site for platelet glycoprotein Ib (GP Ib), a site that has been reported to be distinct from the sulfatide-binding site. In this study, we analyzed the interaction of sulfatides with VWF and its effect on GP Ib-mediated platelet adhesion under flow conditions. Recombinant VWF A1 domain (rVWF-A1) bound specifically and saturably to sulfatides (half-maximal concentration of ∼12.5 µg mL⁻¹), binding that was blocked by dextran sulfate (IC₅₀≈100 µg mL⁻¹) but not by heparin at concentrations up to 100 U mL⁻¹. Furthermore, sulfatides (125 µg mL⁻¹) prevented the adhesion of platelets or glycocalicin-coupled polystyrene beads to a rVWF-A1-coated surface under high shear stress. In addition, plasma VWF prebound to a sulfatide-coated surface failed to support subsequent platelet adhesion. These results provide firm evidence that sulfatides bind the VWF A1 domain at a site overlapping the GP Ib-binding site.     

Assessment of recombinant factor VIIa as an antidote for bleeding induced in the rabbit by low molecular weight heparin

Summary.  While protamine sulfate reverses the anticoagulant effect of standard heparin, there currently is no effective antidote for low molecular weight heparin (LMWH)-induced bleeding. Recently, recombinant activated factor VII (rFVIIa) was approved by the FDA for use in hemophilia patients with factor (F)VIII or FIX inhibitors. However, this new pro-hemostatic agent has potential utility in other clinical scenarios. In this study, we utilized a well-characterized rabbit ear puncture model to test the efficacy of rFVIIa to reverse LMWH-induced prolonged bleeding. Animals were first treated with bolus intravenous LMWH (1800 anti-FXa U kg⁻¹) which increased the primary bleeding time approximately fourfold and raised the plasma anti-FXa activity immediately and continuously throughout the 90-min experiment. In a randomized and blinded fashion, animals then received either rFVIIa (400 µg kg⁻¹) or placebo by bolus intravenous injection, following which the ear puncture bleeding times were measured, along with blood levels of heparin (anti-FXa activity) and FVII. FVII activity increased 5.3-fold over baseline in treated animals, decreasing by only 24% over the full observation period. The rFVIIa-treated animals showed a slight decrease in bleeding time immediately after injection, but there was no statistically significant difference in bleeding after rFVIIa or placebo administration. In this study using a rabbit ear bleeding model, rFVIIa was not an effective antidote to LMWH-induced bleeding. However, the bolus injection of LMWH produced a very high blood anti-FXa level, which may have precluded rFVIIa effectiveness.     

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model

Summary.  Objective: The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 n m , followed by injection of human B domain-deleted FVIII at 180 U kg⁻¹, producing peak plasma concentrations of ∼2 n m . At 2 h, blood loss following a 4-mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti-A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti-C2 inhibitors, (iii) 2–77 and B45, non-classical type II anti-C2 inhibitors, and (iv) 2–117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg⁻¹ overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL⁻¹ and 3 U mL⁻¹ FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose FVIII rather than bypassing agents may be warranted.     

Effects of hemicolectomy on bile acid metabolism in relation to colon carcinogenesis in man

Bile acids are probably important in colon carcinogenesis. Regional differences in bile acid metabolism within the colon were studied to illuminate the preferential distal occurrence of colon cancer in Western countries. Faeces (24 h) were collected for bile acid measurement from 25 patients with hemicolectomy (nine left and 16 right) and 17 adenoma patients with an intact colon (control subjects). Duodenal bile and cytolytic and alkaline phosphatase activity of faecal water were also studied. The median percentage of deoxycholic acid (DCA) was lower in the hemicolectomy groups [left 48% (range 38–57%), right 45% (2–62%) vs. control subjects 59% (38–70%), P  < 0.05]. In duodenal bile, the proportion of DCA in left [4% (1–25%)] was lower than in the patients with right hemicolectomy [19% (0–69%)] and control subjects [24% (7–50%)], P  < 0.05. Faecal concentration of protonated DCA was higher in those with right hemicolectomy (0.101 μmol g⁻¹) than in those with left hemicolectomy (0.048 μmol g⁻¹), which coincided with a higher cytolytic [right 49% (3–93%), left 2% (1–37%)] and alkaline phosphatase activity [right 6.7 U mL⁻¹ (1.2–40.1 U mL⁻¹), left (2.0 U mL⁻¹ (1–25.7 U mL⁻¹), both P  < 0.02]. These findings suggest differences in bile acid metabolism between the proximal and distal colon that may contribute to the disparity in cancer risk.     

Factor XII-dependent increases in thrombin activity induce carboxypeptidase-mediated attenuation of pharmacological fibrinolysis

Summary.  Activation of the contact system in patients treated with fibrinolytic agents may be an important source of thrombin that activates thrombin-activated fibrinolysis inhibitor (TAFI) and attenuates fibrinolysis. Factor (F)XIIa in plasma increased 2-fold over 60 min in patients given either tissue plasminogen activator (t-PA) or streptokinase (SK). To determine whether FXIIa-mediated generation of thrombin and activated TAFI (TAFIa) attenuates fibrinolysis in vitro , plasma clots were incubated with SK (250 U mL⁻¹) or t-PA (2.5 g mL⁻¹) and the rate of lysis was measured. Plasma FXIIa impaired lysis judging from marked acceleration when 2.5 µ m corn trypsin inhibitor were added (lysis increased by 172 ± 144% for SK and 40 ± 31% for t-PA vs. no inhibitor, n  = 16, P  < 0.01). Moreover, inhibition of thrombin with hirudin and TAFIa with carboxypeptidase inhibitor accelerated lysis. We conclude that activation of FXII increases thrombin generation, which promotes TAFIa-mediated attenuation of fibrinolysis.     

Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk

Summary.  Background:  Whether factor XII (FXII) activity, its 46C>T polymorphism and activated FXII (FXIIa) are associated with coronary heart disease (CHD) remains to be determined. Methods:  FXII, FXIIa and the FXII 46C>T polymorphism were determined in a hospital-based cohort of 2615 patients undergoing coronary angiography. Results:  Fifty-seven per cent of the participants were identified as wild-type (46CC), 38% as heterozygous (46CT) and 5% as homozygous (46TT) for FXII 46C>T. FXII and FXIIa levels were significantly lower in carriers of the T-allele: 132 (97–151) U dL⁻¹ FXII in 46CC, 87 (77–99) U dL⁻¹ FXII in 46CT and 53 (42–67) U dL⁻¹ FXII in 46TT carriers ( P  <   0.001), and 2.8 (2.3–3.5) μg L⁻¹ FXIIa in CC, 2.1 (1.6–2.6) μg L⁻¹ FXIIa in CT and 1.2 (0.9–1.5) μg L⁻¹ FXIIa in TT carriers ( P  <   0.001; medians, lower and upper quartiles). Patients with stable CHD ( n  =   935), a history of myocardial infarction ( n  =   785) or who were suffering from acute coronary syndromes (ACS; n  =   323) had significantly lower FXII levels than controls ( n  =   572). The differences remained statistically significant after adjustments for age, sex, diabetes mellitus, smoking, hypercholesterolemia and hypertension. Significantly reduced FXIIa levels in ACS patients lost significance once adjusted for covariates. FXII genotype was not associated with any clinical phenotype. Conclusion:  Lower FXII activity represents an independent risk for CHD and ACS. This is not the case for FXIIa levels or the FXII 46C>T variation.     

SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

Summary.  To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL⁻¹ were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1–29 U dL⁻¹ and 1–33 U dL⁻¹ respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL⁻¹. The median FVIII:C for the third sample was 5.8 U dL⁻¹ with a range of 1.5–36 U dL⁻¹. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL⁻¹; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.     

Evaluation of the safety and pharmacokinetics of a fast-acting recombinant FVIIa analogue, NN1731, in healthy male subjects

Summary.  Background:  NN1731 is a recombinant activated factor VII (rFVIIa) analog with enhanced activity. Objectives:  This clinical trial aimed to assess the safety and pharmacokinetics of single doses of NN1731 in healthy male subjects. Methods : This was a randomized, placebo-controlled dose - escalation trial with four dose tiers (NN1731 5 – 30 μg kg⁻¹). Eight subjects were randomized to either NN1731 ( n  =   6) or placebo ( n  =   2) in each tier. Results:  No thromboembolic or serious adverse events were reported and no antibody formation towards NN1731 was detected. NN1731 was demonstrated to be pharmacologically active based on coagulation-related parameters (prothrombin fragment 1+2, activated partial thromboplastin time and prothrombin time). There were five mild/moderate adverse events in three subjects. The FVIIa activity of NN1731 after ascending single-dose administration of NN1731 fits well with a two-compartment model, indicating a bi-exponential decline with a rapid initial distribution of approximately 73% FVIIa activity (half-life   =   20 min), followed by a less rapid terminal elimination phase eliminating the remaining 27% (half-life   =   3 h). Dose proportionality in healthy male subjects at the dose levels investigated (5 – 30 μg kg⁻¹) was supported by the FVIIa activity data. Conclusions:  Based on the results of this trial, NN1731 appears safe and well tolerated in healthy subjects at doses up to 30 μg kg⁻¹. No immunogenic or thromboembolic events were reported. The pharmacokinetic profile of NN1731 as measured by FVIIa activity appears to follow two-compartment pharmacokinetics characterized by an initial rapid distribution phase followed by a less rapid elimination phase.     

The promoting effect of epinephrine on lipopolysaccharide-induced interleukin-8 production in whole blood may be mediated by thromboxane A2

Summary.  Epinephrine is known to enhance lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion in a platelet dependent manner. To determine whether thromboxane A₂ (TxA₂; a product from activated platelets) is involved in this process, blood samples drawn either before or 2 h after oral administration of 440 mg acetylsalicylic acid (ASA) were stimulated with LPS (5 ng mL⁻¹) and different concentrations of epinephrine were added (0.1–100.0 µmol L⁻¹). ASA ingestion significantly (global P  < 0.05) reduced the enhancing effect of epinephrine on LPS-induced IL-8 release by 15–28%. To further explore whether TxA₂ may be involved in this process, a TxA₂ agonist (U46619) was added to whole blood together with LPS instead of epinephrine. U46619 mimicked the epinephrine effect: 20 ng mL⁻¹ U46619 enhanced LPS-induced IL-8 release by 39% ( P  < 0.05). Furthermore, preincubation of whole blood with 75 µmol L⁻¹ or 150 µmol L⁻¹ SQ29548, a TxA₂ receptor antagonist, completely blocked epinephrine's promoting effect on LPS-induced IL-8 release. Since thrombin-activated platelets have been reported to be important in the production of IL-8 in monocytes through the activation of monocytes by exposed RANTES in a P-selectin-dependent reaction, we suggest that the epinephrine effect is mediated by enhanced TxA₂ production and subsequent rise in the exposure of RANTES and P-selectin on the platelets of whole blood.     

Recombinant factor VIIa reverses the in vitro and ex vivo anticoagulant and profibrinolytic effects of fondaparinux

Summary.  Background : Fondaparinux is a synthetic pentasaccharide, which selectively inhibits coagulation factor (F) Xa, and is registered for prevention of venous thromboembolism following hip fracture, hip replacement, and knee replacement surgery. Recently, it was shown that recombinant FVIIa (rFVIIa) reverses anticoagulant effects of fondaparinux in healthy volunteers. Objectives : In this study, we have explored the in vitro and ex vivo effects of rFVIIa on clot formation and thrombin-activatable fibrinolysis inhibitor (TAFI)-mediated down-regulation of fibrinolysis after fondaparinux administration. Methods :  In vitro clot lysis assays were performed in pooled normal plasma from healthy volunteers to which fondaparinux was added, and in serial samples from healthy volunteers who received a single bolus dose of fondaparinux, a single bolus dose of rFVIIa, or both. Results and conclusions : Fondaparinux significantly delayed clot formation, and clot lysis was significantly increased due to decreased activation of TAFI. Addition of recombinant FVIIa corrected the inhibited clot formation induced by fondaparinux, and the acceleration of clot lysis was partially reversed. In vivo administration of fondaparinux (10 mg) to healthy volunteers similarly resulted in accelerated plasma clot lysis. Subsequent administration of rFVIIa (90 µg kg⁻¹) normalized the clot lysis time up to 6 h postadministration. rFVIIa might be a good therapeutic option in patients treated with fondaparinux who develop bleeding complications, since both clot formation as well as fibrinolytic resistance are improved.     

Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats

Summary.  Background:  Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives:  To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. Methods:  Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results:  rFVIIa (5, 10 and 20 mg kg⁻¹) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg⁻¹ ( n  = 8) and 500 IU kg⁻¹ ( n  = 9), respectively. Similarly, 10 mg kg⁻¹ NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg⁻¹ tinzaparin, 10 mg kg⁻¹ NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg⁻¹ rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions:  This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.     

Increased serum concentrations of the carboxy-terminal cross-linked telopeptide of collagen type I in patients with Gram-negative septicaemia

The authors determined serum levels of the carboxy-terminal cross-linked telopeptide and the carboxy-terminal propeptide of type 1 collagen (ICTP and PICP) in 18 patients with Gramnegative septicaemia before (day 0) and 28 days after therapy and in 18 age- and sex-matched controls by radioimmunoassay. Elevated levels of ICTP were observed in septicaemic patients [median (range): 15 (7–49) μg L⁻¹ before therapy and 14 (6–45) μg L⁻¹ 28 days after therapy vs. 2.1 (1.4–4.3) μg L⁻¹ in normal subjects; P  < 0.01 for both], whereas PICP levels were not different between patients and controls [median (range): 119 (52–275) μg L⁻¹ (day 0) and 133 (79–288) μg L⁻¹ (day 28) vs. 91 (54–213) μg L⁻¹ in normal subjects, P  > 0.05 for all]. The findings suggest an increased production or release of ICTP in Gram-negative septicaemia, presumably owing to an alteration of extracellular matrix during septicaemia-related vascular inflammation.     

Prostaglandin E2 (PGE2) induces headache in healthy subjects

The role of prostanoids in nociception is well established. The headache-eliciting effects of prostaglandin E₂ (PGE₂) and its possible mechanisms have previously not been systematically studied in man. We hypothesized that infusion of PGE₂ might induce headache and vasodilation of cranial vessels. PGE₂ (0.40 µg kg⁻¹ min⁻¹) or saline was infused for 25 min into 11 healthy subjects in a cross-over, double-blind study. Headache intensity was scored on a verbal rating scale from 0 to 10. In addition, we recorded mean flow in the middle cerebral artery (V_MCA) by transcranial Doppler and diameter of the superficial temporal artery (STA) by high-resolution ultrasonography. All 11 subjects reported headache on the PGE₂ day and no subjects reported headache on the placebo day ( P  = 0.001). During the immediate phase (0–30 min) ( P  = 0.005) and the postinfusion phase (30–90 min) ( P  = 0.005), the area under the curve for headache score was significantly larger on the PGE₂ day compared with the placebo day. PGE₂ caused dilatation of the STA (23.5%; 95% CI 14.0, 37.8) and the MCA (8.3%; 95% CI 4.0, 12.6). We suggest that PGE₂ induces headache by activation and sensitization of cranial perivascular sensory afferents.     

Relationship between lipoprotein(a) phenotypes and albumin excretion rate in non-insulin-dependent diabetes mellitus: protective effect of ‘null’ phenotype?

The possible association between lipoprotein(a) [Lp(a)] and albumin excretion rate (AER) is a topic that generates conflicting views. In addition, Lp(a) phenotypes have not previously been considered as factors influencing AER. In order to clarify this issue, we studied 70 non-insulin-dependent diabetes mellitus (NIDDM) patients without clinically detectable macroangiopathy, 27 with microalbuminuria and 43 without it. Both groups were matched for the known variables that could influence AER and serum Lp(a) levels. Lp(a) was determined by enzyme-linked immunosorbent assay (ELISA), and Lp(a) phenotypes were assessed by electrophoresis followed by immunoblotting. Lp(a) phenotypes were grouped as follows: 'small' (F, S1 and S2), 'big' (S3 and S4) and 'null'. The NIDDM patients with microalbuminuria presented higher serum Lp(a) concentrations than the patients without it [15.7 mg dL⁻¹ (95% CI 0.5–36.5) vs. 4.5 mg dL⁻¹ (95% CI 0.1–18.5); P  < 0.001] and a direct correlation between Lp(a) and AER was observed ( r  = 0.34; P  < 0.01). AER was significantly different when Lp(a) phenotypes were considered ['small': median 19 μg min⁻¹ (range 1–195); 'big': median 9.5 μg min⁻¹ (range 1–186); 'null': 4 μg min⁻¹ (range 1–9); P  = 0.04]. None of the NIDDM patients with a 'null' phenotype showed an AER of > 10 μg min⁻¹. In conclusion, this case–control study provides evidence that microalbuminuria is associated with high serum Lp(a) in NIDDM without clinically detectable macroangiopathy. Furthermore, NIDDM patients with a 'null' phenotype could be considered at low risk for the development of microalbuminuria.     

Antistreptokinase platelet-activating antibodies are common and heterogeneous

Summary.  Background : Platelet activation by antistreptokinase (SK) antibodies could impair the clinical effect of SK administration. Objective : To better describe anti-SK antibodies with particular emphasis on procoagulant activities as a result of platelet activation. Patients and methods : Sera were collected from 146 patients with coronary artery disease: non-SK-treated, 95 from mainland France, 31 from French Polynesia; 20 patients from mainland in year 2 after SK treatment. Serum-induced SK-dependent platelet activation resulting in procoagulant activities was assessed with washed platelets from five donors representative of the known patterns of reactivities to IgG. Results : Concentrations (2–5252 µg mL⁻¹) and fibrinolytic neutralization titres (< 10 to > 1280) were found in the expected wide range and correlated (ρ = 0.66, P  < 0.0001). Platelet activation was detected with 145 samples, but varied in intensity and pattern (depending on the donors), although there was no systematic hierarchy; it was presumably due to IgG (inhibited by an IgG Fc receptor-blocking antibody and recovered in the IgG fraction) and only partially affected by aspirin. Marked platelet activation could be detected in samples with concentration as low as 2 µg mL⁻¹, and/or no detectable neutralizing titers. The way of immunization to SK was not found to influence the functional profile of antibodies. Conclusion : Anti-SK platelet-activating antibodies are widespread, heterogeneous, poorly predictable on the basis of their antifibrinolytic effect and strong enough to trigger procoagulant activities. Their clinical relevance should be formally assessed, using patients' own platelets for detection owing to the variation of platelet reactivity.     

rFVIIa and a new enhanced rFVIIa-analogue, NN1731, reduce bleeding in clopidogrel-treated and in thrombocytopenic rats

Summary.  Background: The pharmacological effect of rFVIIa occurs at the surface of activated platelets by enhancing thrombin generation at the site of vascular damage. It is therefore important to study the effects of rFVIIa in platelet-related bleeding situations. We examined the effect of rFVIIa and an rFVIIa-analogue, NN1731, on clopidogrel-induced and thrombocytopenic bleeding in rats. Methods and results: Clopidogrel [10 mg kg⁻¹; per oral (p.o.)] severely inhibited platelet aggregation and increased blood loss after tail-transection four hours after administration. Treatment with rFVIIa (5, 10, 20 mg kg⁻¹) or NN1731 (1, 5, 10 mg  kg⁻¹), administered five minutes after induction of bleeding, reduced blood loss significantly and dose-dependently. NN1731 had an increased hemostatic potential compared with rFVIIa, reducing blood loss to the control level, whereas this was not even achieved with the highest dose of rFVIIa. Antibody-induced thrombocytopenia reduced platelet numbers by more than 90% and increased the blood loss after tail-transection. Treatment with 10 and 20 mg kg⁻¹ rFVIIa significantly reduced blood loss, whereas 10 mg kg⁻¹ NN1731 reduced the bleeding to control levels. Conclusions: The hemostatic effect of rFVIIa and NN1731 was demonstrated in thrombocytopenic and clopidogrel-treated rats, showing efficacy in situations with decreased platelet number or functionality. Our findings are consistent with the hypothesis that rFVIIa/NN1731 contribute to hemostasis by thrombin generation even in situations with platelet disorders. Furthermore, NN1731 demonstrated a higher hemostatic potential than rFVIIa.     

Effect of vascular injury on inhibition of venous thrombosis with ZK-807834, a direct inhibitor of factor Xa

Summary.  Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, K_i 0.11 nM) attenuates progression of thrombosis, but the ED₅₀ is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose–response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023–2.3 µmol kg⁻¹, n  = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED₅₀ was 0.03 µmol kg⁻¹ for non-injured veins and 0.42 µmol kg⁻¹ for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.     

Acenocoumarol therapy in pediatric patients

Summary.  To determine guidelines for administering and monitoring acenocoumarol therapy in children, 93 patients (median 5.1 years, range: 0.2–18 years) were prospectively evaluated over a 33-month period. The loading doses used were: <1 year, 0.20 mg kg⁻¹; >1–5 years, 0.09 mg kg⁻¹; 6–10 years, 0.07 mg kg⁻¹; 11–18 years, 0.06 mg kg⁻¹. In this study, the loading dose and the dose to achieve and maintain target therapeutic range (TTR) for acenocoumarol are age-dependent, with infants having the highest and teenagers having the lowest requirements. The use of a different loading dose according to age has allowed most of the children (80%) in all the age groups to achieve TTR in less than 1 week. No patients had serious bleeding or thrombotic complications. We conclude that there is an age-dependent response to acenocoumarol in pediatric patients. The implementation of an age-adjusted loading dose regimen reduces the length of hospitalization required to achieve effective anticoagulant therapy.     

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome

Summary.  As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL⁻¹ for IgG and/or > 15 U mL⁻¹ for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P  < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL⁻¹) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P = 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.