Heparin induces release of extrinsic: Coagulation pathway inhibitor (EPI)

Extrinsic pathway inhibitor (EPI) is a potent inhibitor of the factor VIIa-tissue thromboplastin complex. The effect of heparin on EPI activity was studied using a chromogenic substrate assay. Addition of heparin to test plasma or whole blood in vitro increased EPI activity. This increase in EPI activity was reduced by the addition of polybrene and/or antibodies to anti-thrombin (anti-AT). Polybrene was therefore added to the assay system. However, part of this effect (up to 20 % of baseline value) was not abolished. After intravenous injection, EPI activity increased dose-dependently. The increase was about 200 % of baseline value after 7500 LJ heparin, and was not reduced by the addition of polybrene and/or anti-AT. A slower and prolonged increase in EPI activity occurred after subcutaneous injections of unfractionated heparin and low molecular weight heparin (LMWH). Venous occlusion failed to increase EPI activity levels. Normal EPI values were observed in patients with severe liver cirrhosis and during warfarin treatment. Gel filtration of human endothelial cell culture supernatant revealed one inhibitory fraction with molecular weight about 43.000. We conclude that EPI probably is produced in endothelial cells and may be released by heparin.     

A sulfated rabbit endothelial cell glycoprotein that inhibits factor VIIa/tissue factor is functionally and immunologically identical to rabbit extrinsic pathway inhibitor (EPI)

Colburn and Buonassisi (In Vitro Cell Dev. Biol. 24, 1133–1136, 1988) have isolated a single chain sulfated glycoprotein inhibitor of factor VIIa/tissue factor-catalyzed activation of factor X from conditioned media of an established rabbit endothelial cell line. We report herein that their endothelial cell-derived inhibitor and extrinsic pathway inhibitor (EPI) isolated from rabbit plasma have identical functional properties with respect to their interactions with factor Xa and with factor VIIa/ tissue factor. In addition, the endothelial cell inhibitor and rabbit plasma EPI migrate with the same apparent molecular weights on non-reduced SDS-PAGE and contain similar amounts of N-linked carbohydrate. Like the endothelial cell inhibitor the EPI of rabbit plasma exists as a single chain molecule. Furthermore, the endothelial cell inhibitor is recognized and neutralized by a polyclonal antibody raised against rabbit plasma EPI. We therefore conclude that cultured rabbit endothelial cells produce an inhibitor of factor VIIa/tissue factor activity that is functionally and immunologically identical to rabbit plasma EPI.     

Effect of a highly selective plasma-kallikrein synthetic inhibitor on contact activation relating to kinin generation, coagulation and fibrinolysis

A highly selective inhibitor of plasma-kallikrein (PK), N-(trans -4-aminomethylcyclohexylcarbonyl)- -phenylalanine 4-carboxymethylanilide hydrochloride, was designed and synthesized by the authors' group, called PKSI-527 in our laboratories. (I) PKSI-527 inhibited PK with a Ki value of 0.81 μM. By contrast, the Ki values for glandular kallikrein(GK), plasmin, thrombin, urokinase and factor Xa were >500 μM, 390 μM, >500 μM, 200 μM and >500 μM, respectively. (II) Effects of PKSI-527 on bradykinin (BK) generation, coagulation and fibrinolysis by contact activation were examined using human plasma. (a) BK generation induced by kaolin appeared to be reduced by PKSI-527. Furthermore BK generation induced by λ-carrageenan, a strong inflammatory agent, was also reduced by PKSI-527. (b) Partial thromboplastin time (PTT) was prolonged by PKSI-527, indicating the suppression of the intrinsic coagulation system. (c) Euglobulin clot lysis time (ECLT) of plasma which was shortened by activation with kaolin, was prolonged by the addition of PKSI-527, confirming the participation of PK in contact-fibrinolysis. These results indicate that PKSI-527 shows great potential in elucidating the significance of PK, and as such deserves further investigation.     

Effects of subcutaneously administered dermatan sulfate (MF 701) on the coagulation and fibrinolytic parameters of healthy volunteers

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was > 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin as compared to placebo. The effect of DS was dose-dependent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PAI and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single s.c. doses of DS (200, or 400 mg) inhibit thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.     

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 ± 5.8%, controls 104 ± 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis β-thromboglobulin (β-TG), thrombinantithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of β-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept.

One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.     

Isolation and anticoagulant properties of a new sulfated xylan: Comparison with heparin and a sodium pentosan polysulfate (SP-54)

Larchwood xylan was purified by diaminoethylaminoethyl (DEAE) cellulose chromatography, sulfated by heating with chlorosulfonic acid -pyridine complex and the sulfated polysaccharide was isolated by epichlorohydrin triethanolamine (ECTEOLA) cellulose chromatography as the sodium salt. It's molecular weight was 25000 and the specific rotation was []_D20–70°. Electrophoretic analysis of the sulfated xylan along with heparin and SP-54 using lithium acetate-agarose technique showed that the charge density of the xylan sulfate was similar to heparin and it moved as a single component in contrast to commercial heparin which resolved into two while SP-54 moved at the same rate as the marker dye. Anticoagulant properties of the sulfated xylan were compared with heparin and SP-54 by studying its effect on the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) using pooled normal human plasma. The sulfated xylan was more effective than SP-54 in delaying coagulation by all the three measurements while it was less active than heparin in inhibiting APTT and PT.     

Heparin and the thrombin inhibitor argatroban enhance fibrinolysis by infused or bolus-injected saruplase (r-scu-PA) in rabbit femoral artery thrombosis

Enhancement by anticoagulation of thrombolysis with infused or bolus-injected saruplase (r-scu-PA) has been studied using heparin and the thrombin inhibitor argatroban. In a rabbit femoral artery thrombosis model infusion of saruplase (3 – 12 mg/kg, 60 min) caused a dose-dependent thrombolysis. Reperfusion rate after infusion of 3 mg/kg saruplase alone was 3/6, reperfusion time 42 ± 3 min and reocclusion rate 2/3; final patency rate at 120 min was 17 %. Combination of 3 mg/kg saruplase with heparin (150 U/kg + 100 U/kg hr i.v.; 5.3-fold PTT-prolongation) resulted in a reperfusion rate of 6/6 after a reperfusion time of 39 ± 7 min; reocclusion rate was 3/6 and final patency rate was 50 %. Argatroban (1 mg/kg + 3 mg/kg.hr i.v.; 2.3-fold PTT prolongation) in combination with saruplase resulted in a reperfusion rate of 6/6 after 26 ± 5 min; no reocclusion occured and final patency rate was 100 % (p < 0.05 vs saruplase alone). Bolus injection of 6 mg/kg saruplase achieved reperfusion in 5/6 arteries after 15 ± 3 min, but reocclusion rate was 4/5; final patency rate was 17 %. Combination of bolus-injected saruplase with heparin resulted in a reperfusion rate of 4/6 after 8 ± 3 min and no reocclusion occured; patency rate was 67 %. With combination of argatroban and bolus-injected saruplase 6/6 arteries were reperfused after 8 ± 3 min; reocclusion was prevented and final patency rate was 100 % (p < 0.05 vs saruplase-bolus alone). Systemic fibrinogenolysis was more pronounced with bolus injection than infusion of saruplase. The results indicate that arterial thrombolysis with saruplase can be enhanced by heparin and the thrombin inhibitor argatroban. The bolus injection of saruplase resulted in persistent reperfusion when simultaneous anticoagulation was performed. Despite less PTT prolongation, enhancement of saruplase-induced thrombolysis was more effective with argatroban than with heparin in rabbit femoral artery thrombosis.     

Studies on Wakan-Yakus (traditional herbal drugs) : Especially on the effects of Gaiyoh (Artemisiae folium) on blood coagulation

Wakan-Yakus (traditional herbal drugs) such as Akyoh (Glutinum), Gaiyoh (Artemisiae folium), Sanshishi (Gardeniae fructus), Kizutsu (Aurantii fructus), and Taisoh (Zizyphi fructus) were studied in relation to their effects on blood coagulation-fibrinolysis. (1) All of the water extracts of the Wakan-Yakus prolonged aPTT and PT. The potency of the effectiveness on aPTT was in the order of Gaiyoh (Artemisiae folium)> Kizutsu (Aurantii fructus)> Sanshishi (gardeniae fructus)> Taisoh (Zizyphi fructus )> Akyoh (Glutium). (2) Gaiyoh (Aremisiae folium)> Kizutsu (Aurantii fructus) Akyoh (glutinum) Taisoh (Zizyphi fructus) showed the antifibrinolytic effects in this order. On the other hand, Sanshishi showed the accelerating effect on fibrinolysis. (3) The inhibition modes of both thrombin and plasmin by Gaiyoh (Artemisiae folium) were shown to be competitive on Lineweaver-Burk plot. (4) Gayoh (Artemisiae folium) was gel-filtrated on Sephadex G-25 column (1.5×90cm) equilibrated with distilled water at room temperature. Five fractions were obtained, and in the first to fourth fraction, strong anticoagulant effects on aPTT and PT were observed. We pooled first and second to make fraction I, and make fraction II from peak 3. The recovery rate was 4.2 % by weight, and 36.7 % by inhibition activity, and specific activity on the basis of inhibition to aPTT was 34.8 % U/mg in the case with fraction II. Fraction I was found to be the same characteristically on blood coagulation. Fraction II was further purified by Sephadex LH-20 column (1.5 × 80 cm) at room temperature. Three fractions (Fraction IIa, IIb, IIc) were obtained, and the strong inhibitory effects was observed on both aPTT and PT in each fraction. The first fraction (fraction IIa) showed the strong inhibitory effect on aPTT, and the heightened specific activity with 17.6 % as the recovery rate. Fraction IIb showed strong inhibitory effect on fibrinolysis. Fraction IIb and IIc were found to be similar with fraction IIa characteristically on blood coagulation-fibrinolysis, but could separate the substance for coagulation and fibrinolysis each other on the basis of weight/volume. (5) by ex vivo study using rabbits, aPTTs were prolonged gradually after infusion (60 mg of fraction IIa). White blood cells and platelets were decreased gradually.     

Clinical evaluation of low-molecular-weight heparin (FR-860) on disseminated intravascular coagulation (DIC) - a multicenter co-operative double-blind trial in comparison with heparin -

This study was evaluated the effectiveness, safety and utility of FR-860 and to compare those with heparin in patients with Disseminated intravascular coagulation (DIC). A diagnosis of DIC was made based on the criteria proposed by the Research Committee on DIC in the Ministry of Health and Welfare of Japan. FR-860 (FR group,75 anti-factor Xa international units/kg/day) and Heparin (HP group, 240 units/kg/day) were administered for 5 days by continuous intravenous infusion. The total number of enrolled patients was 126 cases, and after excluding 1 case a total of 125 cases. Moderate or higher improvement of bleeding symptoms was 33.3% in the FR group and 18.5% in the HP group. On the organic symptoms, FR group showed a significantly higher improvement rate than the HP group, 20.5% and 8.2% respectively. On the overall efficacy of cases with pretreatment plasma AT III levels of less than 21 mg/dl or less than 70%, FR group showed a significantly higher improvement rate than the HP group. The safety rate of FR-860 (93.4%) was a significantly higher than that of the HP group (79.7%). Our report demonstrates that FR-860, as a therapeutic agent for the treatment of patients with DIC, is significantly higher safety and clinical utility as compared with heparin.     

Human pharmacological studies of a defined low molecular weight heparin fraction (Fragminr) evidence for a simultaneous inhibition of factor Xa and IIa (thrombin)

The effect of a low molecular weight heparin (FragminR) (20–80 U/kg) was studied in 10 healthy male volunteers after intravenous administration. Clotting ab-abnormalities are extensive shortly after injection, lasting for 1 – 2 hours depending on heparin dosage. Dilute thrombin time, showing a close correlation to anti-F-Xa activity, is as sensitive as anti-F-Xa. The parallel time course of dilute thrombin time and anti-F-Xa activity clearly demonstrates that LMWH has an effect on F-IIa lasting as long as that of F-Xa. After subcutaneous administration clotting abnormalities are only slight with 40–60 U/kg, in contrast to the intravenous study. The platelet system remains unchanged.     

Heparin-induced platelet aggregation: Dose/response relationships for a low molecular weight heparin derivative (pk 10169) and its subfractions

Addition of heparin or heparin derivatives to citrate anticoagulated platelet-rich plasma caused platelet aggregation in a dose-dependent manner. Utilizing heparin, a low molecular weight heparin derivative (PK 10169) and its various subfractions, we determined dose/response relationships for platelet aggregation and found that the ability of these agents to cause platelet aggregation was dependent upon the molecular weight of the individual subfraction used. In comparison to unmodified porcine mucosal heparin, the lower molecular weight derivative (PK 10169) yielded a dose/response curve that was shifted down and to the right, and indicated that this agent was less potent in causing platelet aggregation. In addition, as the molecular weight of PK 10169 subfractions decreased, their dose/response curves were progressively shifted down and to the right. The lowest molecular weight subfraction was essentially without platelet aggregating activity. We also measured the anti IIa and anti Xa activities of these agents and concluded that these activities did not appear to correlate with platelet aggregating activity. Platelet aggregation studies with PK 10169 subfractions of high and low affinity for antithrombin III (AT III) indicated that the platelet aggregating activity of these compounds may not be related to their affinity for AT III, but results were not definitive.     

Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa

We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a K_d in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP-and thrombin-stimulated gel filtered platelets caused a 2–3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.     

Inhibition of red blood cell-induced platelet aggregation in whole blood by a nonionic surfactant, poloxamer 188 (Rheothrx Injection)

RheothRx Injection, an aqueous solution of a nonionic block copolymer (poloxamer 188) formulated for intravenous administration, was investigated as an inhibitor of red blood cell (RBC)-induced platelet aggregation at plasma concentrations of 0.05-5mgmL⁻¹. Platelet aggregation was determined by measuring the fall in single platelet counts after mechanical agitation of 2mL aliquots of citrated whole blood in a 37°C shaking waterbath. Inhibition of RBC-induced platelet aggregation of >95% was observed for poloxamer 188 at a concentration of 1mgmL⁻¹, and 41% inhibition was observed at 0.05mgmL⁻¹. Poloxamer 188 was observed to be a more effective inhibitor of RBC-induced platelet aggregation than 2-chloradenosine (2-ClAd) or phosphoenolpyruvate/pyruvate kinse (PEP/PK). Studies using platelet rich plasma (PRP) showed that platelet aggregation could not be induced by shaking in the absence of RBC, though aggregation was induced by the addition of exogenous adenosine diphosphate (ADP). Poloxamer 188 did not inhibit ADP-induced platelet aggregation. We propose that poloxamer 188 protects RBC from mechanical trauma by non-specific adsorption of copolymer to the RBC surface (via the hydrophobic polyoxypropylene moiety), and that this effect prevents mechanical damage and hence leakage of ADP from RBC. RheothRx Injection has been shown to have value in the treatment of acute ischemic disorders such as myocardial infarction. The observation of significant inhibition of RBC-induced platelet aggregation at clinically relevant concentrations suggests that RheothRx Injection may have antithrombotic properties in vivo, and may therefore have potential not only in acute ischemia but also to prevent thrombosis within vascular prostheses or to prevent rethrombosis after angioplasty or endarterectomy.     

Alpha-fodrin is cleaved by caspase-3 in a chronic ocular hypertensive (COH) rat model of glaucoma

Purpose: α-Fodrin is a neuronal cytoskeletal protein and a known caspase-3 target. We sought to determine whether caspase-3 cleaves α-fodrin in COH rat retinas and whether this process is reduced by adeno-associated virus (AAV)-induced retinal ganglion cell expression of baculovirus inhibitory repeat-containing 4 (BIRC4), a potent caspase-3 inhibitor.Methods: Ocular hypertension was induced unilaterally in five rat eyes by limbal injection of hypertonic saline. In a similar experiment, ocular hypertension was induced in four eyes pre-treated with an intravitreal injection of AAV-BIRC4 to assess α-fodrin cleavage. Western immunoblotting was performed on all retinas.Results: Caspase-3 cleavage of α-fodrin yields a specific 120 kDa protein fragment. COH retina immunoblots indicated significantly more caspase-3 cleavage of α-fodrin than controls (P<0.01, paired t-test). Inhibition of retinal caspase-3 activity with BIRC4 reduced caspase-3-mediated α-fodrin cleavage compared to controls.Conclusion: This confirms our previous finding of caspase-3 cleavage of α-fodrin in COH retinas and parallels pathology seen in Alzheimer’s disease, in which neurons undergo chronic caspase activation, slow build-up of cleavage products, and delayed apoptosis. If caspase activation in glaucoma leads to protracted rather than rapid retinal ganglion cell apoptosis, a much longer therapeutic window exists for apoptosis inhibition with caspase inhibitors such as BIRC4.     

Kinetic study on the initial stage of the fibrinogen-fibrin conversion by thrombin (I) application of mathematical treatment to turbidimetrical method

Kinetics on the initial reaction of the fibrinogen-fibrin conversion catalyzed by thrombin was studied by turbidimetrical measurement. The turbidity change caused by the conversion reaction was analyzed by a mathematical treatment, assuming that the dimer formation of fibrin monomers was the rate-determining reaction in the stage of polymerization. The obtained relations were investigated under experimental conditions at neutral pH and at low ionic strength, in which fast polymerization rate of fibrin monomers can be expected in comparison with the rate of enzyme reaction. The experimental results analyzed from turbidity-time curves were generally in agreement with theoretical prediction. On the other hand, the plots relating to the enzyme concentration did not lead to anticipated linear relationship. Hence, it was concluded that every fibrin monomer did not always polymerize via dimer formation, but that dimers of fibrin monomers behaved as nuclei for polymerization.     

Avoidance learning in the crayfish (Procambarus clarkii) depends on the predatory imminence of the unconditioned stimulus: a behavior systems approach to learning in invertebrates

Signaled avoidance learning in crayfish (Procambarus clarkii) was investigated when the crayfish were not confined, by indexing two types of locomotive movement to the escape compartment. Mild shocks, which induced tail flipping in the crayfish, and light illumination were used as unconditioned and conditioned stimuli, respectively. In Experiment 1, two groups of crayfish were trained in a one-way shuttle box. The crayfish in Group Forward were placed in the start compartment facing the escape compartment and they were able to escape/avoid shocks by walking forward, while the crayfish in Group Backward were placed in the compartment facing the opposite direction and they were able to escape by tail flipping. Avoidance learning was achieved only by walking, and not by tail flipping despite the fact that consistent tail flipping allowed the crayfish to avoid shocks. In Experiment 2, the experimental conditions were switched by using the ABA design. In this experiment, we confirmed that avoidance behavior was restricted to walking. These results are readily explained by the behavior systems approach.