The Intercept Blood System Affects Platelet Function and Survival By Inducing Apoptosis

Abstract Introduction The Intercept Blood System (IBS) is currently approved in 40 countries to prevent transfusion-transmitted diseases. It is mandatory in Switzerland since 2012. Our aim was to study the effects of this treatment on platelet function and survival and to investigate the underlying mechanisms. Methods 10 platelet apheresis units (AU) from healthy volunteers were left untreated (non-IBS) or treated with the IBS following standard blood center procedure and storage at 22°C under agitation. Samples were analyzed for aggregation, P-selectin exposure, integrin activation, PS/PE exposure, platelet aggregation over collagen and adhesion to vWF at day (d) 1, 5, 7 and 10. Platelet survival was analyzed in NOD-SCID mice after 1 day storage by injection of fluorescently-labeled non-IBS or IBS platelets and analysis of blood at 0.5/2/5 hours post-injection. Bak expression was analyzed in platelet lysates by Western blotting. Results IBS treatment substantially reduced platelet aggregation already after 1 day storage (area under the curve -AUC- for thrombin 45.3 non-IBS vs 26.7 IBS, p=0.02, fig. A; AUC for collagen 19.1 non-IBS vs 4.9 IBS, p=0.07). P-selectin exposure and activation of a2bb3 were not different between non-IBS and IBS samples, as well as phosphatidylserine exposure as measured by Annexin V binding. Adhesion to vWF under high shear flow was reduced after 1 day storage, albeit not significantly due to some variability (platelet-covered area 4950 um2 non-IBS vs 1481 um2 IBS, p=0.2). Aggregation on collagen under flow was again markedly reduced upon IBS treatment at day 1 and 10 of storage (area d1: 67974 um2 non-IBS vs 35490 um2 IBS, p=0.15; d10: 37761 um2 non-IBS vs 14033 um2 IBS, p=0.13. AUC: 511426 non-IBS vs 247750 IBS, p=0.08). Platelet survival was reduced in NOD-SCID mice injected with IBS platelets compared to non-IBS platelets (% platelets in blood 2h post-injection: 45.5% non-IBS vs 25.1% IBS; 5h: 30.35% non-IBS vs 8.87% IBS; n=3, fig. B). Fluorescent platelets were quantified in spleen sections and found to be significantly higher in mice injected with IBS platelets (average platelet area: 19083 um2 non-IBS vs. 79395 um2 non-IBS, n=3, p=0.03). Analysis of Bak expression in platelet lysates revealed an increased amount of this pro-apoptotic mediator in IBS samples (Bak/tubulin integrated intensities: 0.08 non-IBS vs 0.12 IBS, p=0.007, fig. C), while there was no difference in the anti-apoptotic Bcl-XL. Conclusions The IBS reduces platelet aggregation and responsiveness to physiological agonists in vitro, and platelet survival in the circulation of immunodeficient mice in vivo. This effect might be clinically relevant for patients receiving platelet transfusion and is possibly due to the induction of pro-apoptotic signaling in platelets by the method, which warrants further investigation. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Platelet Function Studies in Normal Volunteers and Patients with Angina Pectoris

10.1055/s-0039-1684612 ◽

1979 ◽

Cited By ~ 1

Author(s):

J.J.C. Jonker ◽

den G.J.H. Ottolander

Keyword(s):

Platelet Aggregation ◽

Angina Pectoris ◽

Platelet Function ◽

Normal Range ◽

Double Blind ◽

Group Iii ◽

Platelet Survival ◽

Group I ◽

Group Ii

In 30 normal subjects (group I) and in 89 patients with angina pectoris we studied: the platelet survival time (PST), the platelet aggregation test I (PAT I) acc. to Breddin, the platelet aggregation ratio (PAR) acc. to Wu and Hoak and the Filtragometer log TA acc. to Hornstra. The patients were divided in two groups: 46 patients had already been treated for 6 months with Clofibrate (group II) and 43 patients with placebo (group III) in a double blind trial. The average PST (T½) was within the normal range (group I 99 hrs. group II 105,7 hrs.; group III 102,0 hrs.). About 20% of patients of group II and III had abnormally shortened T½. The PAT I was on average abnormal in group II and III (PAT I in group II 2,3; group III 2,7), but group II normalized after 12 months treatment (PAT I 1,85). The PAR was abnormal in group III, while group II was within the normal range (group I 0,87; group II 0,82; group III 0,69). The log TA results were abnormal in group II and III (group I 2, 45, group II 2,1; group III 2, 1), after 12 months treatment the patient group remained abnormal (group II 2,2; group III 2,1). We failed to find a correlation between the four platelet function tests, nor with these tests and basic laboratory values. The PAT I, the PAR and the Filtragometer seems to be valuable in the detection of abnormal platelet behavior in vitro, but it does not mean than an abnormal platelet survival in vivo occurs in the same individuals.

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In Vitro Characterization of Platelet Dysfunction In Common Hematological Disorders Using the Verify Now® Assay

Blood ◽

10.1182/blood.v116.21.1436.1436 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1436-1436

Author(s):

Nina D’Abreo ◽

Vatsala Kirtani ◽

Jennifer L. Kujawa Eisenstein ◽

Yelda Nouri ◽

Matt Geller ◽

...

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Reference Ranges ◽

Platelet Dysfunction ◽

Platelet Defects ◽

Verify Now ◽

Instructions For Use

Abstract Abstract 1436 Objectives: Myelodysplastic syndrome (MDS), immune thrombocytopenia purpura (ITP) and myeloproliferative disorders (MPD) are frequent diagnoses in a community hematology practice. Qualitative platelet defects are common features of these disorders but are difficult to characterize in daily practice as specialized technology is not readily available. The Verify Now® (VN) is a rapid, point-of service assay to measure platelet aggregation. VN assesses the degree of inhibition of GPIIb/IIIa mediated aggregation and uses minimal sample preparation. Its main application currently, is to determine the adequacy of anti-platelet therapy with aspirin (ASA), thienopyridines, such as clopidogrel (CPL), and GPIIb/IIIa inhibitors in patients with cardiovascular disease. (Verify Now for IIb/IIIa, aspirin, and P2Y12, Instructions for Use, Accumetrics) We used VN to detect platelet aggregation defects in patients with ITP, MDS and MPD and compared the to a platelet function analyzer PFA -100. (Francis JL; In Michelson AD, ed. Platelets, 2nded. San Diego: Elsevier/Academic Press, 2007:535-544) Methods: Subjects with MDS, ITP, and MPD were identified from our Hematology practice. Informed consent was obtained from all patients. Patients taking aspirin and /or clopidogrel were not excluded. Platelet function was determined concurrently using VN and PFA-100 using standard reference ranges. (Verify Now for IIb/IIIa, aspirin, and P2Y12, Instructions for Use, Accumetrics), (Mammen EF et al; Semin Throm Hemost. 1998; 24 (2):195-205). Results: Thirty three patients are enrolled in the study, 21 with ITP, 6 with MDS and 6 with MPD. Assay results are available for 30 patients. Platelet counts for all patients ranged from 2 to 1206 ×103/cmm, mean platelet count being 184 ×103/cmm (SD ± 215) and median count was 120 ×103/cmm. ITP: In 15 patients with ITP and platelet count ≥ 100 ×103/cmm, 11 were not on ASA/CPL. PFA-100 and VN each detected abnormalities in 4 patients but results were non-concordant in 2 patients. In 4 patients on ASA, VN detected ASA effect in all 4 whereas the PFA-100 detected 2. In 2 patients with platelet count < 100 ×103/cmm not on ASA/CPL, VN detected abnormalities in both, whereas PFA detected only 1. (Table 1) MDS: Two patients had counts ≥ 100 ×103/cmm. One was on ASA and both assays detected ASA effect. Neither assay detected an abnormality in the patient not on ASA. In 4 patients with counts <100 ×103/cmm, 3 were not on ASA/ CPL. Both assays detected abnormalities in all 3 and ASA effect in 1 patient on ASA. (Table 2) MPD: In 4 patients with counts < 400 ×103/cmm, not on ASA/CPL, both assays detected non-concordant abnormalities in 1 patient. Both assays detected CPL and ASA effect in 1 patient taking both drugs. 2 patients had count ≥400,000. In 1 patient taking CPL, VN detected CPL effect but PFA did not. In 1 patient on ASA, PFA detected abnormality but VN did not. (Table 3) Conclusion: In patients with MDS, ITP, and MPD, abnormal platelet function is common. These results show that VN may be as sensitive for detecting platelet dysfunction as PFA-100. Results were especially concordant in MDS patients. We plan to include 150 patients to confirm these results. We hope to define qualitative platelet defects in this population so that appropriate intervention can be recommended prior to invasive procedures. Disclosures: No relevant conflicts of interest to declare.

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A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649933 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1316-1322 ◽

Cited By ~ 13

Author(s):

Mary Ann McLane ◽

Jagadeesh Gabbeta ◽

A Koneti Rao ◽

Lucia Beviglia ◽

Robert A Lazarus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Sequence Similarity ◽

Platelet Aggregation Inhibitors ◽

Antiplatelet Drug ◽

Rgd Sequence ◽

Fibrinogen Receptor ◽

Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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The Effect of Dimethyl Sulfoxide on In Vitro Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648027 ◽

1976 ◽

Vol 36 (01) ◽

pp. 221-229 ◽

Cited By ~ 16

Author(s):

Charles A. Schiffer ◽

Caroline L. Whitaker ◽

Morton Schmukler ◽

Joseph Aisner ◽

Steven L. Hilbert

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Dimethyl Sulfoxide ◽

Platelet Function ◽

Platelet Volume ◽

Short Term ◽

Platelet Factor ◽

Short Term Exposure ◽

Structural Abnormalities

SummaryAlthough dimethyl sulfoxide (DMSO) has been used extensively as a cryopreservative for platelets there are few studies dealing with the effect of DMSO on platelet function. Using techniques similar to those employed in platelet cryopreservation platelets were incubated with final concentrations of 2-10% DMSO at 25° C. After exposure to 5 and 10% DMSO platelets remained discoid and electron micrographs revealed no structural abnormalities. There was no significant change in platelet count. In terms of injury to platelet membranes, there was no increased availability of platelet factor-3 or leakage of nucleotides, 5 hydroxytryptamine (5HT) or glycosidases with final DMSO concentrations of 2.5, 5 and 10% DMSO. Thrombin stimulated nucleotide and 5HT release was reduced by 10% DMSO. Impairment of thrombin induced glycosidase release was noted at lower DMSO concentrations and was dose related. Similarly, aggregation to ADP was progressively impaired at DMSO concentrations from 1-5% and was dose related. After the platelets exposed to DMSO were washed, however, aggregation and release returned to control values. Platelet aggregation by epinephrine was also inhibited by DMSO and this could not be corrected by washing the platelets. DMSO-plasma solutions are hypertonic but only minimal increases in platelet volume (at 10% DMSO) could be detected. Shrinkage of platelets was seen with hypertonic solutions of sodium chloride or sucrose suggesting that the rapid transmembrane passage of DMSO prevented significant shifts of water. These studies demonstrate that there are minimal irreversible alterations in in vitro platelet function after short-term exposure to DMSO.

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The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649080 ◽

1973 ◽

Vol 30 (02) ◽

pp. 315-326

Author(s):

J. Heinz Joist ◽

Jean-Pierre Cazenave ◽

J. Fraser Mustard

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Barbituric Acid ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Primary Response ◽

Sodium Pentobarbital ◽

Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

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Interactions of Staphylokinase with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653799 ◽

1995 ◽

Vol 73 (03) ◽

pp. 472-477 ◽

Cited By ~ 5

Author(s):

H R Lijnen ◽

B Van Hoef ◽

D Collen

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Specific Binding ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Normal Plasma ◽

Atp Secretion ◽

Platelet Disaggregation ◽

Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657151 ◽

1982 ◽

Vol 47 (02) ◽

pp. 150-153 ◽

Cited By ~ 13

Author(s):

P Han ◽

C Boatwright ◽

N G Ardlie

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Antiarrhythmic Drugs ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Second Phase ◽

Ionophore A23187 ◽

High Concentrations ◽

Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

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Effects of Ticlopidine of Platelet Function in Men with Stable Angina Pectoris

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660138 ◽

1985 ◽

Vol 54 (04) ◽

pp. 808-812 ◽

Cited By ~ 7

Author(s):

Ulf Berglund ◽

Henning von Schenck ◽

Lars Wallentin

Keyword(s):

Platelet Aggregation ◽

Angina Pectoris ◽

Platelet Function ◽

Plasma Levels ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Controlled Study ◽

Double Blind ◽

Platelet Factor

SummaryThe effects of ticlopidine (T) (500 mg daily) on platelet function were investigated in a double-blind placebo-controlled study in 38 middle-aged men with stable incapacitating angina pectoris. The in vitro platelet reactivity to aggregating agents, the platelet sensitivity to prostacyclin and the plasma levels of platelet specific proteins and fibrinogen were determined before and after 4 and 8 weeks of treatment. T exerted a potent inhibitory effect on ADP- and collagen-induced platelet aggregation. The effect of T was proportional to the pretreatment reactivity to ADP and collagen. The inhibitory effect of T on the epinephrine response was less pronounced. The plasma levels of beta-thromboglobulin, platelet factor 4 and fibrinogen were not influenced by T. The platelet inhibition of prostacyclin was potentiated by T, and it was demonstrated that T and prostacyclin had synergistic inhibitory effects on platelet aggregation.

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Modulation of platelet function by reactive oxygen metabolites

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1994.267.1.h308 ◽

1994 ◽

Vol 267 (1) ◽

pp. H308-H318 ◽

Cited By ~ 2

Author(s):

G. Ambrosio ◽

P. Golino ◽

I. Pascucci ◽

M. Rosolowsky ◽

W. B. Campbell ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Guanylate Cyclase ◽

Reactive Oxygen Metabolites ◽

Cyclic Monophosphate ◽

Cgmp Formation ◽

Oxygen Metabolites ◽

Exogenous H2o2

Reactive oxygen metabolites have been reported to affect platelet aggregation. However, this phenomenon is still poorly understood. In the present study we investigated the effects of superoxide radical and hydrogen peroxide (H2O2) on platelet function in vitro and correlated those effects to possible changes of platelet concentrations of cyclic nucleotides and thromboxane, since these systems play a key role in the response of platelets to activating stimuli. Human platelets were exposed to xanthine-xanthine oxidase (X-XO), a system that generates both superoxide radicals and H2O2. Sixty seconds of incubation with X-XO impaired aggregation in response to ADP (by 48%), collagen (by 71%), or the thromboxane mimetic U-46619 (by 50%). This effect was reversible and occurred in the absence of cell damage. Impairment of aggregation in platelets exposed to X-XO was due to H2O2 formation, since it was prevented by catalase but not by superoxide dismutase. Similarly, incubation with the pure H2O2 generator glucose-glucose oxidase also markedly inhibited ADP-induced platelet aggregation in a dose-dependent fashion. Impaired aggregation by H2O2 was accompanied by a > 10-fold increase in platelet concentrations of guanosine 3',5'-cyclic monophosphate (cGMP), whereas adenosine 3',5'-cyclic monophosphate levels remained unchanged. The inhibitory role of increased cGMP formation was confirmed by the finding that H2O2-induced impairment of platelet aggregation was largely abolished when guanylate cyclase activation was prevented by incubating platelets with the guanylate cyclase inhibitor, LY-83583. Different effects were observed when arachidonic acid was used to stimulate platelets. Exposure to a source of H2O2 did not affect aggregation to arachidonate. Furthermore, in the absence of exogenous H2O2, incubation with catalase, which had no effects on platelet response to ADP, collagen, or U-46619, virtually abolished platelet aggregation and markedly reduced thromboxane B2 production (to 44% of control) when arachidonic acid was used as a stimulus. In conclusion, our data demonstrate that H2O2 may exert complex effects on platelet function in vitro. Low levels of endogenous H2O2 seem to be required to promote thromboxane synthesis and aggregation in response to arachidonic acid. In contrast, exposure to larger (but not toxic) concentrations of exogenous H2O2 may inhibit aggregation to several agonists via stimulation of guanylate cyclase and increased cGMP formation.

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Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

Thrombosis and Haemostasis ◽

10.1160/th13-11-0919 ◽

2014 ◽

Vol 112 (08) ◽

pp. 412-418 ◽

Cited By ~ 15

Author(s):

Nima Vaezzadeh ◽

Ran Ni ◽

Paul Y. Kim ◽

Jeffrey I. Weitz ◽

Peter L. Gross

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Saphenous Vein ◽

Ex Vivo ◽

Arterial Injury ◽

Inhibitory Antibody ◽

Study Objective ◽

Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

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