Pharmacokinetics and pharmacodynamic effects of a new antibody glycoprotein IIb/IIIa inhibitor (YM337) in healthy subjects.

BACKGROUND: This study describes the first administration of YM337, the Fab fragment of a humanized monoclonal antibody against the fibrinogen GP IIb/IIIa receptor, in healthy male humans. METHODS AND RESULTS: Platelet aggregation (20 micromol/L ADP), platelet adhesion, fibrinogen binding, bleeding time, and YM337 concentrations in plasma were studied in substudy 1 after single boluses of 0.025, 0. 05, 0.1, 0.2, and 0.4 mg/kg YM337 and in substudy 2 after a bolus (0. 35 mg/kg) plus 6 hours of infusion at different dose levels of YM337 (0.5, 0.75, 1.0, 1.5 microg. kg(-1) x min(-1)), with abciximab as reference drug (n=5 or 6 subjects per group). After the 0.2-mg/kg and 0.4-mg/kg boluses, fibrinogen binding was reduced by >80% and bleeding time was prolonged to approximately 60 minutes. Bolus followed by infusion of 1.0 and 1.5 microg x kg(-1) x min(-1) YM337 maintained inhibition of platelet aggregation >80%. Aggregation and bleeding time returned to normal within 24 hours. A bolus of 0.25 mg/kg of abciximab followed by an infusion of 0.125 microg x kg(-1) x min(-1) showed effects similar to those observed with the 0.5- and 0. 75-microg x kg(-1) x min(-1) infusion of YM337. In 53 subjects exposed to YM337, 1 case of transient thrombocytopenia and 3 minor bleeding events occurred. No human anti-chimeric antibodies were detected 2 weeks and 2 months after administration. CONCLUSIONS: YM337 effectively inhibits IIb/IIIa-mediated platelet aggregation and adhesion in humans. The results of this phase 1 study will give rise to further clinical evaluation of YM337.

Thrombophilia and venous thromboembolism. International consensus statement. Guidelines according to scientific evidence.

Thrombophilia is the term now used to describe predisposition to increased risk of venous and occasionally arterial thromboembolism due to hematological abnormalities. It can be a multifactorial disorder where congenital defects of anticoagulant or procoagulant factors may be combined with acquired hematological abnormalities. It should be considered in patients with a documented unexplained thrombotic episode or a positive family history. The aim of this document is to provide guidelines for investigation and management of patients with thrombophilia in the presence or absence of venous thromboembolism (VTE).

Ex vivo--in vitro interaction between aspirin, clopidogrel, and the glycoprotein IIb/IIIa inhibitors abciximab and SR121566A.

OBJECTIVES: To assess the interaction between aspirin and clopidogrel in healthy male volunteers and the interaction of the glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors abciximab and SR121566A with blood from those pretreated subjects (ex vivo-in vitro). METHODS: Aspirin (300 mg/day), clopidogrel (75 mg/day), or the combination of both drugs were administered orally for 8 days. Group 1 (n = 5) started with aspirin and group 2 (n = 5) with clopidogrel. From day 4 to day 8, subjects of both groups received the combined treatment. Blood from these subjects was spiked with abciximab (0.5 and 1.5 microg x mL(-1)) and SR121566A (31 and 62 ng x mL(-1)). RESULTS: In vivo, average bleeding times were 6.8 minutes at baseline, 20.3 minutes for clopidogrel alone (P < .01), 10.9 minutes for aspirin alone (difference not significant), and 24.0 minutes (P < .01) for the combined treatment. Fibrinogen binding to the platelet GPIIb/IIIa receptor was reduced for aspirin to 69% (difference not significant), to 63% for clopidogrel (difference not significant), and to 63% for the clopidogrel plus aspirin combination (P < .01). CD62 expression as a marker of platelet granular secretion was reduced to 66% by clopidogrel (P < .01) and to 41% by the combination of clopidogrel and aspirin; aspirin alone had no effect. In vitro, with pretreatment with aspirin and clopidogrel, inhibitory effects of the GPIIb/IIIa inhibitors on fibrinogen binding were additive to changes observed with aspirin or clopidogrel alone. No effect on CD62 expression was observed with either GPIIb/IIIa inhibitor. Aspirin and clopidogrel reinforced effects of the GPIIb/IIIa inhibitors on adenosine diphosphate (5 micromol/L)-induced aggregation in an additive manner, a supra-additive effect was observed with collagen (2 microg x mL(-1))-induced aggregation. CONCLUSION: The augmentation of the antiaggregatory effects of GPIIb/IIIa inhibitors by aspirin and clopidogrel and the lack of antisecretory effects of GPIIb/IIIa inhibitors may favor their combination with clopidogrel.

Spontaneous platelet aggregation as a predictive risk factor for vascular occlusions in healthy volunteers? Results of the HAPARG Study. Haemostatic parameters as risk factors in healthy volunteers.

The HAPARG Study (haemostatic parameters as risk factors in healthy volunteers) was performed in a subset of volunteers taking part in the MARISK Study (Mainzer Risikoindikatoren Studie für die koronare Herzkrankheit) sponsored by the German Ministry of Research and started in 1984. A previous study (Yamanishi et al., Thromb Haemostas 1985;54:539-543) had shown that spontaneously enhanced platelet aggregation as measured with the PAT-III-test and higher fibrinogen concentrations are significant risk factors for new vascular occlusions in diabetic patients. It was the aim of the HAPARG Study to establish whether spontaneous platelet aggregation and other hemostatic variables are independent risk factors for vascular occlusions in healthy volunteers. Employees of a chemical/pharmaceutical company aged 40-65 years and personnel of the University of Mainz, aged 30-60 years were included in this prospective study. Besides anamnestic data such as on smoking, hypertension and diabetes, blood pressure, the ankle/arm Doppler-index and an ECG were recorded and serum cholesterol, HDL, LDL, triglycerides, uric acid and glucose were measured. Men (1884) and women (989) entered the study and were followed for 4-6 years. In the age group of 30-50 years, more women than men were included. During the observation period 53 vascular occlusions occurred (36 coronary and nine cerebral events and eight peripheral vascular occlusions). Only three of these endpoints occurred in women. Besides age (odds ratio = 1.7, P = 0.02) and gender as expected risk factors, the multivariate logistic stepwise regression analysis revealed smoking (odds ratio = 2.2, P = 0.008), lower HDL-levels (odds ratio = 2.2, P = 0.013), elevated diastolic blood pressure (odds ratio = 1.4. P = 0.004) followed by spontaneous platelet aggregation (odds ratio = 1.1, P = 0.037), and slightly elevated blood glucose (P = 0.0047) as significant risk factors for men. Higher fibrinogen levels missed significance in this analysis (P = 0.059). None of the other hemostatic parameters showed a significant correlation with the vascular events. To our knowledge, this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. Spontaneously enhanced platelet aggregation is probably an independent risk factor and, like elevated fibrinogen and other haemostatic variables, an indicator of an ongoing active atherosclerotic process.

Effects of acetylsalicylic acid on human platelet function in vivo at salicylate steady state.

The results of clinical trials concerning the use of acetylsalicylic acid (ASA) as antithrombotic drug are contradictory. Inhibition by ASA of platelet prostaglandin synthesis and aggregation is prevented by its metabolite salicylic acid (SA) in animals and in human platelets in vitro. It was suggested that ASA might produce its own inhibitor, thereby diminishing its efficiency in thromboembolic disease. In four healthy male subjects there was no difference in inhibition of collagen-induced platelet aggregation after the administration of 500 mg ASA alone or at salicylate steady state (3 g SA daily). But the inhibition of tissue-extract-induced platelet shape change was diminished and shortened by pretreatment with SA. We conclude that SA does not inhibit the effects of ASA on human platelet aggregation in vivo in therapeutic dose ranges. The clinical importance of the SA/ASA-interaction on tissue-extract-induced platelet shape change remains to be clarified.

Treatment of patients with peripheral arterial occlusive disease Fontaine stage IV with intravenous iloprost and PGE1: a randomized open controlled study.

In a randomized open controlled study the clinical effects and tolerability of prostaglandin E1 (PGE1) and the stable prostacyclin (PGI2) analogue, iloprost in the management of diabetic and non-diabetic patients with advanced peripheral arterial occlusive disease (PAOD Fontaine stage IV) were compared. 267 patients were enrolled in this multicentre study and treated for 21-28 days, either by daily infusions of 6 h with iloprost or 2 x 2 h with PGE1. At the end of treatment patients were assessed for evidence of improvement of trophic lesions, relief of rest pain and change of global clinical status. 228 patients were considered as evaluable for efficacy analysis, which revealed 52.7% responders in the iloprost group and 43.1% for PGE1 (p = 0.148). Whereas iloprost showed similar effects in diabetics and non-diabetics (53.3% and 51.4% response rates, respectively), the diabetics treated with PGE1 had a considerably poorer outcome (36.6% versus 53.3%). At 6 months follow-up 62.2% of patients in both groups were alive with a viable limb. Slightly more iloprost patients underwent major amputation (32.1% versus 27.2%), but the number of deaths was reduced by 50% in the iloprost group compared to the PGE1 group (7.5% versus 14.6%, p = 0.10). Side-effects such as headache, flushing and gastrointestinal symptoms were significantly more common in the iloprost group (73.9%) than in the PGE1 group (31.0%), particularly during the first 3 days of dose titration. No specific toxic or unexpected reactions were reported in either group.

Coronary heart disease, unstable angina, PTCA: new indications for low molecular weight heparins?

Unstable angina in patients with coronary heart disease carries a high risk of myocardial infarction (MI) and death. Partial thrombotic occlusions of coronary arteries are a major causal factor in the development of unstable angina and consecutive thrombotic occlusions may lead to MI. Treatment with unfractionated heparin (UFH) has been shown to significantly reduce the frequency of anginal attacks, but not to affect the frequency of MI, PTCA and death within one month. Prolonged treatment with low molecular weight heparins (LMWHs) may improve the outcome in patients with unstable angina. New clinical studies in this area seem promising. In the prevention of early reocclusions after PTCA LMWHs may be more effective than UFH. Late restenosis after PTCA within 6 months occurs in 25-35% of patients. To date, no effective prophylaxis exists. In a recent trial, short-term treatment with a LMWH had no preventive effect on late restenoses. Ongoing trials with long-term treatment may lead to better results. In the near future long-term treatment, up to six months, with a LMWH alone or in combination with a platelet function inhibitor, possibly with reduced doses of both drugs, may reduce the frequency of restenosis.

Anticoagulant and antithrombotic effects of combinations of defibrotide with heparins and other antithrombotic agents in an animal thrombosis model.

Defibrotide, acetylsalicylic acid, low molecular weight heparin (CY 216), sodium heparin, Molsidomin and a thromboxane receptor antagonist have been investigated separately and in combinations in an animal thrombosis model in which rat mesenteric venules are damaged by defined laser energy. Furthermore, the ex vivo anticoagulant activity of Defibrotide and heparin were studied in rat plasma. All investigated agents showed a significant and dose-dependent antithrombotic effect when venules were damaged. A strong additive effect was observed when Defibrotide was administered together with heparins. The combinations of Defibrotide with other antithrombotic agents did not have significant additive effects. In the present study the combination of Defibrotide and heparins showed a prolongation of aPTT and Heptest in comparison with heparin given exclusively.

Changes of spontaneous and induced platelet aggregation in the presence of a human endothelial cell monolayer.

A new, simple method has been developed for the investigation of platelet aggregation in the presence of a cultured confluent human endothelial cell monolayer, in disk shaped rotating cuvettes, without magnetic stirring. The endothelial cells inhibited the aggregating effect of several inducers in a concentration dependent manner. At a platelet count of 5 x 10(5)/microliter in PRP e.g. the aggregating effect of 1 microgram/microliter thrombin was completely abolished. Spontaneous aggregation was also prevented by the EC-monolayer. A correlation could be established between the inhibitory effect of ECs and the number of platelets. In PRP with a platelet count of 7 x 10(-5)/microliters the inhibitory effect of the endothelial cell monolayer significantly decreased.

Antiplatelet and anticoagulant effects of "HN-11 500," a selective thromboxane receptor antagonist.

The antiplatelet and anticoagulant effect of a thromboxane receptor (TX receptor) antagonist developed by Nycomed (Linz) has been studied in a placebo-controlled double-blind phase I study. Sixteen healthy male volunteers received different single oral doses of "HN-11 500" (C(14)H(15)NO(5)S(2); 1, 10, 100, 200, and 400 mg). Eight volunteers received placebo. The washout period between each dosage applied was at least 12 days. Platelet aggregation induced by the thromboxane mimetic "U 46 619" (C(21)H(34)0(4)) and platelet adhesion to siliconized glass were significantly and dose-dependently inhibited. The effect lasted between 3 and 4 h (10 mg) and 8 h (400 mg), respectively, and correlated well with the pharmacokinetic data. Platelet aggregation seems to be more sensitive to monitor the effects of HN-11 500 on platelet function than platelet adhesion. Plasma levels of 300 ng/ml HN-11 500 probably leads to >90% inhibition of platelet aggregation. The template bleeding time slightly increased but did not exceed the normal range. Furthermore, there was a wide variation of results. There were no significant changes in platelet counts, platelet-induced thrombin generation time (PITT), and blood coagulation parameters. All doses of HN-11 500 were well tolerated. HN-11 500 is a potent TX receptor antagonist (TXRA), which inhibits either platelet aggregation or platelet adhesion, which has not yet been described. In clinical routine, TXRAs have to demonstrate the effectiveness in large clinical trials for different clinical indications and to compete with single or combined administrations of cyclooxygenase (COX) inhibitors, thienovridines, thromboxane synthase inhibitors, and GIIb/IIIa inhibitors.

Functional studies on platelets of a patient with an acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIB/IIIA complex.

Platelet functions have been studied of a 63 year old woman with a severe acquired thrombopathy. The platelets did not adhere to siliconized glass. Aggregation could not be induced by either ADP (1 microM) nor collagen (2 micrograms/ml), no release of serotonin was found under these conditions. Thrombin caused only a weak aggregation response. Quantitative analysis of platelet actin revealed a very low total actin content (473 micrograms/10(9) platelets) and an extremely low F-actin value (3% of total actin). Stimulation of platelets with 0.1 U/ml thrombin for 3 min resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Ca2+ movement in the patient's platelets is severely impaired after ADP and collagen stimulation, whereas a normal Ca2+ movement was obtained by 0.1 U/ml thrombin. The inhibition of the functions of normal platelets (aggregation and actin polymerization) by addition of patient's serum (5-10% final concentration) points to receptor blockade by platelet autoantibodies in the patient's serum. The antibody was purified by adsorption on Protein-A-Sepharose. Addition of IgG-suspension (5% final concentration) to washed control platelets resulted in similar effects on aggregation and actin polymerization compared to the effects of patient's serum.

Platelet membrane defects in fawn hooded bleeder rats.

An inbred strain of fawn hooded rats with a congenital platelet defect shows a marked bleeding tendency with prolonged bleeding time. This haemorrhagic disorder has been exclusively related to a deficiency of nucleotides in platelet dense granules. When tested in cell electrophoresis platelets from fawn hooded bleeder rats showed a significantly lower electrophoretic mobility than normal rat platelets. Subsequent studies on the platelet membrane protein pattern by high resolution two-dimensional gel electrophoresis revealed the deficiency of a membrane glycoprotein (apparent molecular mass 90.000, isoelectric point 5.6), which is detectable in normal rat platelets after surface labeling by periodate-tritiated sodium borohydride. It seems likely, that this glycoprotein defect contributes at least partially to the disorder of platelet function in fawn hooded bleeder rats.

Regression of deep vein thrombosis by i.v.-administration of a low molecular weight heparin--results of a pilot study.

Twenty-five patients with phlebographically confirmed deep vein thrombosis of the lower limb were treated with intravenous infusions of low molecular weight heparin for 7 to 29 days. The mean dosage was 15.2 +/- 3.0 Uanti-Xa (equivalent 7.6 +/- 1.5 U-aPTT). Phlebograms were taken before, during and after the treatment with low molecular weight heparin and evaluated using the score system of Marder. Nearly complete recanalization of the occluded veins was found in six (24%) patients, improvement of the Marder score of 60 to 90% was found in four patients and of 30 to 60% in seven patients, while eight patients remained unchanged. With an average dose of 15.2 I.U./kg/h the heptest was prolonged to 70 to 120 seconds while the aPTT-level did not significantly increase. tPA-antigen-levels increased significantly in most of the patients after the third day of treatment, while PAI-activity remained unchanged. A positive conclusion between the decrease of the Marder score and the duration of treatment was found. Thus the low molecular weight heparin used in this investigation proved to be effective and safe in treating deep vein thrombosis.

Platelet activation and its role in thrombin generation in platelet-induced thrombin generation time.

Platelet-induced thrombin generation time (PITT) is a newly developed global coagulation assay in which a small amount of partially anticoagulated platelet-rich plasma (PRP) is rotated in a disc-shaped cuvette within the light beam of a photometer. The time intervals from onset of rotation until aggregation and coagulation of the sample are registered. The aim of our study was to compare platelet activation with generation of thrombin during rotation of PRP in PITT system. Aliquots of PRP were taken before, 1, 3, and 8 min after the onset of rotation as well as at the beginning of aggregation and shortly before coagulation. Thrombin activity was measured with chromogenic substrate S-2238. We have also measured the level of generated prothrombin activation fragment 1+2 (F1+2), which reflects the concentration of liberated thrombin. Platelet activation was assayed by means of platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) concentration and registration of the aggregation. The concentrations of the F1+2, PF4, beta-TG increased very slowly from the beginning of the test until aggregation occurred. From the start of aggregation, the levels of F1+2 rose rapidly. In contrast to the F1+2 measurements, thrombin activity has not been detected from onset of rotation until the end of the test. Only trace thrombin activity was detectable just after the plasma sample had been clotted in the cuvette. Our results demonstrate that there exists a close relationship between platelet activation and thrombin generation. Viable platelets, which adhered to the cuvette walls, form an active template on which thrombin can be generated from prothrombin.

Differential in vitro effects of the platelet glycoprotein IIb/IIIa inhibitors abixicimab or SR121566A on platelet aggregation, fibrinogen binding and platelet secretory parameters.

The aim of this study was to compare fibrinogen binding, inhibition of platelet aggregation and secretory potential of the MAb abciximab (0.5-5 microg/mL) and the peptidomimetic compound SR121566A (15-250 ng/mL) in vitro in whole blood. Fibrinogen binding was followed by flow cytometry; platelet function was evaluated by light transmittance and by impedance aggregometry. Secretory functions of platelets were evaluated using ATP as marker for early secretion by dense granulae and P-selectin (CD62) for alpha-granular secretion as well as CD63 for lysosomal degranulation. Results showed that fibrinogen binding induced by 5 microM TRAP was maximally inhibited greater than 80% at 3 microg/mL abciximab or at 250 ng/mL SR121566A. At these concentrations of antagonists, platelet aggregation induced by 5 microM ADP or 2 microg/mL collagen was inhibited completely. Expression of CD62 was reduced 34% with abciximab or 15% with SR121566A; CD63 expression was reduced 22% with both agents. With both agents, the EC50 for inhibition of CD62 and CD63 expressions was in similar magnitudes than the EC50 for fibrinogen binding inhibition. With 3 microg/mL abciximab, ATP secretion was maximally reduced to 50% of the control, whereas SR121566A at 250 ng/mL had no inhibitory effect on this parameter. A slight increase in ATP secretion was seen with 0.5 microg/mL abciximab and with SR121566A in concentrations of less than 45 ng/mL. The data suggest a discoupling between the anti-aggregatory and the antisecretory effects of IIb/IIIa antagonists. Because it is not established to what extend CD62 or CD63 expression can be reduced by any means, the reduction by 20-30% obtained by 3 microg/mL abciximab or 250 ng/mL SR121566A might already be the maximum possible inhibition by these agents.

In vitro dose response to different GPIIb/IIIa-antagonists: inter-laboratory comparison of various platelet function tests.

AIMS: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration-response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). METHODS: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 microg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose-response curves were established on the basis of a sigmoidal Imax)-model [I=(Imax)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50) 0.84 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/ml). A similar pattern was observed for abciximab. CONCLUSIONS: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the "real" inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.

Laboratory monitoring of new antithrombotic drugs.

This article examines laboratory methods that are used or may be used to monitor newly developed heparins, low-dose vitamin K antagonists, new thrombin inhibitors such as recombinant hirudin, and oligopeptide thrombin inhibitors (some of which it is hoped will be orally active), thromboxane receptor antagonists, glycoprotein IIb-IIIa inhibitors, and ticlopidine-like structures.

Low molecular weight heparins--state-of-the-art and unsolved issues.

Low molecular weight heparins are well established in the prophylaxis of deep vein thrombosis in patients with general surgery, in high risk patients undergoing elective hip surgery or emergency surgery and also in patients with an enhanced risk of thrombosis who are treated in medical wards. There are, however, many possibilities for improving prophylaxis and treatment with LMWH. The mechanisms by which low molecular weight heparins and also unfractionated heparin inhibit thrombus formation are not fully understood. The inhibition of thrombin formation and local effects at the endothelial level may be more important than antithrombin-III mediated effects on factor IIa and on factor Xa. For most low molecular weight heparins the most effective dose regimens to be used in patients at high risk have not yet been established. Low molecular weight heparins may be more effective in the treatment of deep venous thrombosis than unfractionated heparin. In the therapeutic studies published so far the major intention was to show that low molecular weight heparins can prevent the progression of deep venous thrombosis and pulmonary embolism to the same extent as unfractionated heparin. Extended treatment regimens, however, may lead to a relevant thrombus reduction. Outpatient treatment for a longer period of time with results not far from those obtained with thrombolysis seem possible especially in elderly patients. Low molecular weight heparins in their present form or modified low molecular weight heparins may be useful for long-term treatment of patients with atherosclerosis with the aim of regression of atherosclerotic lesions. New forms of application, e.g. inhalation, may render long-term treatment more feasible.

A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

Experimental studies on the anticoagulant and antithrombotic effects of sodium and calcium pentosan polysulphate.

In the present study we have compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulphate (Na-PPS, Ca-PPS). The antithrombotic effect of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules diameter of 20-30 microm were injured by well defined Argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities (aPTT, Heptest) of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses above 10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS an antithrombotic effect was not observed. Oral application of Ca-PPS in doses higher than 20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose 10 mg/kg, the aPTT increased 3-fold and Heptest 2.5-fold compared to controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS no effect on coagulation test could be measured.