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.     

Effect of heparin on in vitro platelet reactivity in cardiac surgical patients-a comparative assessment by whole blood platelet aggregometry and haemostatometry

The effect of heparin on platelet reactivity was assessed simultaneously by haemostatometry (response to shear stress) and whole blood platelet aggregometry response to collagen (WBPA). From each blood sample a ratio (H_R for haemostatometry and M_R and I for WBPA) showing platelet reactivity in the presence or absence of heparin (5 U/ml) was calculated. A value < 1 represented a proaggregatory effect and > 1 an inhibitory effect. Non-anticoagulated blood samples obtained from 290 cardiac surgical patients were tested by haemostatometry and citrated whole blood samples from 100 patients with aggregometry.

Haemostatometry demonstrated a proaggregatory effect of heparin in 8.6% (25) and an inhibitory effect in 91.4% (265). Assessed by WBPA, heparin was proaggregatory in 41–46% and inhibitory in 54–59%.

In the 100 patients tested by both methods there was a significant correlation between the findings with the two techniques (r = 0.46, p < 0.0001).

A wide individual variation in the platelet effect of heparin was demonstrated. This variation appeared greater and a higher proportion showed inhibition when blood was tested by haemostatometry.     

Inhibition of human platelet function in vitro and ex vivo by acetaminophen

The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, ¹⁴C-5HT secretion, and thromboxane B₂ (TxB₂) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB₂ formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB₂ formation was inhibited by 40–99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB₂ formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion m 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB₂ formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with ³H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD₂ and PGE₂, as well as TxB₂. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro.     

Monoclonal antibodies to bovine platelet factor 4: Species interactions to platelets and megakaryocytes using indirect immunocytofluorescence

Murine monoclonal antibodies (mAb) were raised to a purified product of bovine PF-4, a 9,500 dalton protein with heparin neutralization activity comparable to that of human PF-4. Using a non-radioactive slide immunoenzymatic assay, four major classes of mAb could be identified when comparisons were made between purified antigens of PF-4 and β-TG-like protein from both bovine and human species. Type 1 cross-reacted with all four antigens; type 2 reacted with PF-4s; type 3 reacted with only bovine PF-4 and β-TG-like protein; and type 4 reacted only with bovine PF-4. Differences in immunoreactivities of types 1, 2 and 3 were retained throughout the growth of succeeding clones and in ascitic fluids. Using a modified factor Xa, S-2222 chromogenic substrate-heparin inhibition assay, no mAb was found to block PF-4's ability to neutralize heparin. mAbs representative of types 1, 2 and 3 were successfully raised in stable cell lines from at least second generation clones. These were purified with protein A agarose and found to be IgG₁. By indirect immunocytofluorescence a purified type 2 mAb, 2E7, was found to specifically stain granules of human platelets and megakaryocytes, as well as masses (putative platelets within late stage megakaryocytes) without staining other cellular types in either bone marrow or peripheral blood. Species comparisons displayed positive staining for human, rat, and rabbit platelets and megakaryocytes, and negative staining for mouse, guinea pig and dog platelets and megakaryocytes. It seems likely that mAb, 2E7, is directed against an epitope, common to PF-4 of bovine, human, rabbit and rat.     

Sex-related differences in platelet morphology in whole blood (WB) and platelet-rich plasma (PRP)

The proportion of smooth, disc-shaped platelets (D) in freshly drawn, glutaraldehyde fixed whole blood (WB) and citrated platelet-rich plasma prepared at 37°C is compared for male (N = 55) and female (N = 31) donors. Female donors have significantly more %D than male donors and the variability in %D measured on repeated occasions over a period of 3–69 months is less for female donors. WB and PRP gave similar results. The sex-related differences in %D were not related to hematocrit or to concomitant use of oral contraceptives. There were no significant sex-related differences in platelet mean volume or in platelet plasma membrane surface area as determined by the osmotic spherocyte method. However, the volume of D is smaller for male donors. It is concluded that the sex-related differences in platelet morphology do not represent intrinsic differences in platelet size or measurable total plasma membrane but represent a selective shape change activation of the larger D in the circulation of male donors. The significance of these observations for the sex-related differences in risk of cardiovascular disease and efficacy of anti-thrombotic therapy awaits appropriate prospective epidemiological studies.     

Evidence that plasma fibrinogen and platelet membrane GPIIb-IIIa are involved in the adhesion of platelets to an artificial surface exposed to plasma

We investigated the molecular mechanism(s) by which platelets adhere to an artificial surface exposed to plasma, using polystyrene microtiter plates pretreated with plasma. Washed platelets labelled with ⁵¹Cr were incubated with the plates under static conditions. Prostaglandin E₁(PGE₁) was added to the platelets to prevent platelet-platelet interactions. Adhesion required the presence of a divalent cation such as Mg⁺⁺ or Ca⁺⁺. Polyclonal anti-fibrinogen antibody inhibited adhesion by 70%. Polyclonal antibodies against fibronectin, vitronectin, von Willebrand's Factor, and the Fc portion of human IgG, had no effect on adhesion. Platelets adhered normally to a surface pretreated with plasma from a patient with severe von Willebrand's disease. No platelet adhesion occurred when the surface was pretreated with an afibrinogenemic plasma. Monoclonal antibodies against platelet membrane GPIIb-IIIa, potent inhibitors of ADP-induced fibrinogen binding to platelets, completely inhibited adhesion. Monoclonal antibodies against the GPIb subunit and GPIc(VLA ₅) showed no inhibitory effects on adhesion. Platelets from a patient with Glanzmann's thrombasthenia (type 1) did not adhere to the surface pretreated with normal plasma. These results suggest that plasma fibrinogen adsorbed onto the surface and that platelet membrane glycoprotein(GP)IIb-IIIa were responsible for adhesion in an activation-independent manner.     

A comparison of the effects of three positive inotropic agents (amrinone, milrinone and medorinone) on platelet aggregation in human whole blood

Amrinone, milrinone and medorinone inhibit platelet aggregation in human whole blood. They are particularly potent inhibitors of arachidonic acid induced aggregation, inhibiting by 50% (IC₅₀) at concentrations of 1.5μM (milrinone), 7.5μM (medorinone) and 48μM (amrinone). Each drug was less potent at inhibiting ADP and collagen-induced aggregation. The rank order for inhibition of arachidonic acid - induced aggregation correlated well with the rank order of cyclic AMP phosphodiesterase inhibition for these drugs when coupared to the response of a reference cAMP phosphodiesterase inhibitor (CI-930) and a reference cAMP phosphodiesterase inhibitor (M & B 22948). Since inhibition of platelet aggregation occurred at clinically relevant concentrations, it is evident that these agents have potentially beneficial antithrombotic properties.     

Quantitation of platelet deposition on human arteries: Assessment of the disparity between results obtained with indium (in)-labelling versus scanning electron microscopy

Quantitation techniques for measuring platelet deposition (PD) to vessel surfaces are important to an understanding of thrombogenesis. In previous studies, scanning electron microscopy (SEM) has been shown to indicate a lower extent of PD than platelet ¹¹¹In-scintigraphy. Part of this disparity may be explained by nonspecific binding of ¹¹¹In to the vessel surface during perfusion, or loss of adherent ¹¹¹In-labelled platelets by lysis or dissociation from the surface during specimen preparation for SEM. To assess whether these independent processes occur, we used a previously described human placental artery (HPA) perfusion model to quantify vessel ¹¹¹In retention. Of the total ¹¹¹In that bound to the vessel surface during perfusion, 77 ± 42% (N=9) was platelet associated ¹¹¹In (¹¹¹In-labelled platelets) and 23 ± 19% (N=9) was non-platelet associated ¹¹¹In (nonspecific binding). After specimen fixation, 67 ± 32% (N=9) of the initial total surface ¹¹¹In remained. This decrease is due to dissociation of both adherent ¹¹¹In-labelled platelets, and non-platelet associated ¹¹¹In. After fixation, 57 ± 34% (N=9) of the initial total surface ¹¹¹In remained as ¹¹¹In-labelled platelets and 10 ± 13% (N=9) remained as non-platelet associated ¹¹¹In. Fixation caused no measurable lysis of platelets. These data suggest that PD may be overestimated by ¹¹¹In-scintigraphy because of nonspecific binding of ¹¹¹In and underestimated by SEM because of dissociation of adherent platelets during specimen preparation for SEM.     

Clinical relevance of the fibrinogen uptake test in patients undergoing elective general abdominal surgery—relation to major thromboembolism and mortality

Postoperative thromboembolic complications were evaluated in 2578 patients undergoing elective abdominal surgery, all receiving prophylaxis with low molecular weight heparin. A positive fibrinogen uptake test (FUT) developed in 217 patients (8.4%), while 37 patients (1.4%) had major thromboembolism (TE, defined as proximal deep vein thrombosis and/or pulmonary embolism, verified with phlebography, pulmonary scintigraphy or autopsy). In only 14% a positive FUT was associated with a major TE event. In 19% of the patients with major TE the FUT was negative. In multiple logistic regression the independent predictors for major TE were partially different from those for positive FUT. Thirty day mortality was 3.0%. There were significant associations between both positive FUT and major TE on one hand and mortality on the other (relative risks 2.4 and 5.8, respectively). FUT is not a good predictor of major TE. Both positive FUT and major TE indicate a significant risk of postoperative death.     

Differential involvement of membrane (Na-K)atpase in thrombin- and trypsin-mediated platelet activation

The possibility that platelet activation may also involve membrane (Na-K)ATPase was investigated by testing the effects of both proteinases on platelet shape change and aggregation in the absence and presence of the specific (Na-K)ATPase inhibitor ouabain. Ouabain (8 to 80 μM) completely antagonized trypsin-induced platelet shape change and aggregation when it was preincubated with platelet suspension before the addition of trypsin. Unlike trypsin, thrombin-induced platelet activation was significantly enhanced by ouabain. It was also observed that on partially purified beef heart (Na-K)ATPase preparation, thrombin significantly enhanced the enzyme inhibition caused by submaximal inhibitory concentrations of ouabain.

Soybean trypsin inhibitor (4 μg/ml) employed as the agent capable to counteract proteinase effects on the (Na-K)ATPase, was shown both to prevent and antagonize the platelet activation induced by trypsin (0.3 to 1.5 μg/ml), but it failed to modify the responses evoked by thrombin.

It is concluded that membrane (Na-K)ATPase is involved differently in platelet activation by trypsin and thrombin probably because receptor sites to which either proteinase on the platelet surface binds, are distinct. Direct enzyme involvement is indeed apparent only in trypsin-induced platelet activation.     

Effects of different anticoagulants on human platelet size distribution and serotonin (5-HT) induced shape change and uptake kinetics

The effect of collecting blood with disodium ethylene diamine tetra-acetate (EDTA), citrate (NAC) or acid sodium citrate-dextrose (ACD) as anticoagulants on platelet count and size distribution was investigated. No difference between the three preparations regarding platelet count was found in whole blood. Preparation of platelet-rich plasma (PRP) significantly reduced the platelet count in NAC-PRP (p<0.01) to a value of 288 × 10⁹/1 compared to those of 365 × 10⁹/1 and 368 × 10⁹/1 in EDTA and ACD blood respectively. A significant shift in the platelet size in EDTA-PRP towards larger cell volumes was observed. There were no differences in the size distribution patterr between NAC-PRP and ACD-PRP in spite of the differences in platelet count. Platelet 5-HT uptake kinetics in EDTA-PRP showed a 50 per cent reduction in both K_m and V_max compared to that in ACD-PRP. The study shows that the receptor mediating 5-HT induced shape change has a direct opposite pH dependence than that of the 5-HT carrier. Interference of receptor-mediated responses in 5-HT uptake studies in human platelets is clearly minimized at a lowered pH. The finding is probably of importance in disorders associated with platelet hyperaggregability.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

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.     

Influence of thromboembolism prophylaxis by low molecular weight heparin CY 216 (fraxiparine®) on several parameters of haemostasis in patients under dialysis and receiving either unfractionated heparin or CY 216

The hemorragic risk of an association of the low molecular weight (LMWH), Fraxiparine® injected intravenously at the dose of 7.500 AXaICU or of unfractionated heparin (UFH) injected intravenously at the usual dose used during hemodialysis (3.750 ± 1.280 IU + 1.000 IU after 2 hours of dialysis) to the subcutaneous administration once daily of a thromboembolism preventive dose of Fraxiparine (7.500 AXaICU) was evaluated on the modification of the following hemostasis parameters : thrombin time, activated partial thromboplastin time (APTT), anti Xa activity, in 13 uremic patients on hemodialysis. The association of intravenous and subcutaneous Fraxiparine prevented efficiently the clotting of the extracorporeal circulation without inducing a detectable antithrombinic activity. In contrast, the association of I.V. UFH to subcutaneous Fraxiparine induced a significant increase of the thrombin time and of the APTT, so explained by the activity of UFH. It is concluded that subcutaneous Fraxiparine at the thromboembolism preventive dose can be associated as well to I.V. Fraxiparine as to UFH without increasing the potential hemorrhagic risk. Nevertheless the association of SC and IV Fraxiparine 7 500 AXaIC u seems preferable to the association of SC Fraxiparine with UFH.     

New insights in heparin-induced thrombocytopenia by the use of fluid-phase assays to detect specifically platelet factor 4/heparin complex antibodies and antibody-secreting cells

Introduction

The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required.

Materials and Methods

We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin.

Results

The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab.

Conclusion

The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.     

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.     

Aqueous extracts of Ocimum basilicum L. (sweet basil) decrease platelet aggregation induced by ADP and thrombin in vitro and rats arterio--venous shunt thrombosis in vivo

Objective

To study the effects of aqueous extract of Ocimum basilicum L (OBL) on platelet aggregation and experimental thrombus.

Methods

Platelet aggregation induced by ADP (5 μM) and thrombin (4 UI), and thrombus weight in an arteriovenous thrombosis (AVT) model were tested after 2 weeks treatment with 15, 75 and 375 mg/kg OBL orally in rats, compared to 8.8 mg/kg/day aspirin. AVT was also tested 2 h after 75 mg/kg OBL orally, after 3 and 7 days treatment, and one, three and seven days after the end of a two-week treatment. Analysis was done by ANOVA followed by protected t-tests (Tukey).

Results

OBL (15, 75, 375 mg/Kg) dose-dependently inhibits platelet aggregation by ADP and thrombin, with 75 mg/kg/day having approximately the same effect as 8.8 mg/kg/day aspirin. ADP induced aggregation reached 45%, 28% and 18% for OBL, respectively, 15, 75, 375 mg/kg compared to 71% for control and 27% for aspirin (all p < 0.01 except aspirin vs. OBL 75 mg/kg/day p = 0.7). Thrombin-induced aggregation reached 33%, 22%, 21% for OBL, respectively, 15, 75, 375 mg/kg compared to 67% for control and 48% for aspirin (all p < 0.01 except OBL 75 vs. OBL 375 mg/kg/day, p = 1.0). Compared to a control thrombus weight of 48.1 mg (SD 4.9), thrombus weight was 29.4 (3.3), 19.0 (1.9) and 12.3 (1.7) after treatment for 2 weeks with 15, 75 and 375 mg/kg OBL, respectively, and 27.4 (5.3) after 8.8 mg/kg aspirin (all p < 0.001 except aspirin vs. OBL 75 mg/kg/day p = 1.0). Maximum effect of OBL was reached after one week's treatment. The effect subsided between 3 and 7 days.

Conclusion

OBL possesses an inhibitory effect on platelet aggregation induced by ADP and thrombin, that is dose-dependent and results in an anti-thrombotic effect in vivo which develops progressively over 7 days and disappears over 3-7 days. The active ingredient now needs to be characterized.