Platelet Hyperreactivity to Submaximal Epinephrine: Biologic and Genetic Correlates.

Abstract Platelet hyperreactivity constitutes an important thrombotic risk factor; however, standardized methods for its measurement are lacking. We recently reported that aggregometry using a submaximal concentration of epinephrine (0.4 μM in citrated platelet-rich plasma) identifies individuals with in vitro platelet hyperreactivity; this hyperreactivity was reproducible on multiple occasions over long periods of time (up to 3 years). To better understand this aberrant reactivity, we studied in a larger group of subjects (n=404) the association between healthy individuals’ platelet reactivity to epinephrine and their platelet phenotype as measured by other functional and biochemical assays. Fourteen percent (n=56) of our study cohort showed a hyperreactive response (> 60% aggregation) to 0.4 μM epinephrine; the remainder showed a minimal response (see figure). Subjects with hyperreactivity to epinephrine were more likely to exhibit hyperaggregability to other agonists (ADP, arachidonic acid, collagen, collagen-related peptide and ristocetin; p<.04), increased spontaneous aggregation (p<.001), shorter PFA-100 closure times with both epinephrine (p<.001) and ADP (p<.02) cartridges and increased surface expression of P-selectin after exposure to ADP (p<.02) and to shear stress (p<.01). Subjects exhibiting platelet hyperreactivity in citrated specimens also did so in specimens anticoagulated with the thrombin inhibitor Phe-Pro-Arg-chloromethyl ketone, suggesting that the hyperreactive phenotype is independent of calcium concentration and residual plasma thrombin activity. Taken together, these data obtained using multiple assays and methods of platelet stimulation are most consistent with the existence of a hyperreactive phenotype that is not agonist specific, but is rather a "global" property of the platelet. Platelet hyperreactivity was not associated with age, race, body mass index, smoking status or presence of hypertension, but was independently associated with female gender (p=.02) and with higher fibrinogen levels (p=.002). To explore potential mechanisms responsible for platelet hyperreactivity to epinephrine, we measured expression of key surface membrane glycoproteins (GPs) and genotyped polymorphisms located on candidate genes relevant to epinephrine-mediated platelet activation. We found that hyperreactivity was strongly associated with increased expression of both quiescent and activated forms of the fibrinogen receptor GP IIb-IIIa (p<.005) and with the T allele of the C825T polymorphism on the gene (GNB3) encoding the beta-3 subunit of G proteins (p<.03), but not with the G1838A polymorphism on the gene encoding the α2A-adrenergic receptor. Aggregometry using submaximal concentrations of epinephrine thus identifies a global hyperreactive platelet phenotype and may be useful in settings where platelet hyperreactivity bears special relevance - for example, in populations of patients at risk for thrombosis. Our findings also suggest that the physiologic determinants of platelet hyperreactivity in response to different types of stimulation may share common signaling pathways, possibly involving G protein-coupled receptors and increased GP IIb-IIIa expression. Variation in Platelet Reactivity (404 healthy subjects) Variation in Platelet Reactivity (404 healthy subjects)

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Reaction of Acetaldehyde with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648393 ◽

1992 ◽

Vol 67 (01) ◽

pp. 126-130 ◽

Cited By ~ 8

Author(s):

Olivier Spertini ◽

Jacques Hauert ◽

Fedor Bachmann

Keyword(s):

Platelet Aggregation ◽

Inhibitory Action ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Shape Change ◽

Reaction Medium ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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Norepinephrine, but not epinephrine, enhances platelet reactivity and coagulation after exercise in humans

Journal of Applied Physiology ◽

10.1152/jappl.1999.86.1.133 ◽

1999 ◽

Vol 86 (1) ◽

pp. 133-138 ◽

Cited By ~ 53

Author(s):

Hideo Ikarugi ◽

Tomomi Taka ◽

Shoko Nakajima ◽

Takanori Noguchi ◽

Sadahiro Watanabe ◽

...

Keyword(s):

Aerobic Exercise ◽

Resting State ◽

Platelet Reactivity ◽

Healthy Male ◽

Thrombotic Risk ◽

In Vitro Method ◽

Plug Formation ◽

Exercise In Humans ◽

Catecholamine Levels

The effects of exercise and catecholamines on platelet reactivity or coagulation and fibrinolysis appear to be inconsistent. This may be partly due to the methods employed in previous studies. In the present study, we investigated the effects of acute aerobic exercise and catecholamines on the thrombotic status by a novel in vitro method, shear-induced hemostatic plug formation (hemostatometry), using nonanticoagulated (native) blood. Aerobic exercise (60% maximal O2consumption) was performed by healthy male volunteers for 20 min, and the effect on platelet reactivity and coagulation was assessed by performing hemostatometry before and immediately after exercise. Exercise significantly increased shear-induced platelet reactivity, coagulation, and catecholamine levels. The effect of catecholamines on platelet reactivity and coagulation was assessed in vitro by adding catecholamines to blood collected in the resting state. The main findings of the present study are that elevation of circulating norepinephrine at levels that are attained during exercise causes platelet hyperreactivity and a platelet-mediated enhanced coagulation. This may be a mechanism of an association of aerobic exercise with thrombotic risk.

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Further characterization of platelet-type von Willebrand's disease in Japan

Blood ◽

10.1182/blood.v64.6.1254.1254 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1254-1262 ◽

Cited By ~ 25

Author(s):

H Takahashi ◽

M Handa ◽

K Watanabe ◽

Y Ando ◽

R Nagayama ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Sodium Dodecyl ◽

Membrane Glycoproteins ◽

Glycoprotein Ib ◽

Normal Platelet ◽

Platelet Receptor ◽

Von Willebrand ◽

Willebrand Factor

Abstract We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D- arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.

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A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽

10.1182/blood.v83.3.677.bloodjournal833677 ◽

1994 ◽

Vol 83 (3) ◽

pp. 677-682 ◽

Cited By ~ 1

Author(s):

WX Li ◽

AV Kaplan ◽

GW Grant ◽

JJ Toole ◽

LL Leung

Keyword(s):

Ex Vivo ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Shear Rates ◽

High Shear Rates ◽

Dependent Inhibition ◽

Platelet Thrombus

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

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Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽

10.1182/blood.v68.4.927.bloodjournal684927 ◽

1986 ◽

Vol 68 (4) ◽

pp. 927-937

Author(s):

FM LaDuca ◽

RE Bettigole ◽

WR Bell ◽

EB Robson

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Platelet Membrane ◽

Type I ◽

Membrane Glycoproteins ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

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PLATELET FUNCTION AND PLASMA FIBRINOGEN IN YOUNG SURVIVORS OF MYOCARDIAL INFARCTION

10.1055/s-0038-1643019 ◽

1987 ◽

Author(s):

U Berglund ◽

H von Schenk ◽

L Wallentin

Keyword(s):

Myocardial Infarction ◽

Platelet Function ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Plasma Fibrinogen ◽

Women Patients ◽

Platelet Factor ◽

Young Survivors

An increased liability for thrombosis might be of pathogenetic importance in young survivors of myocardial infarction (MI). In 73 (58 men and 15 women) patients with MI below 45 years of age and 73 matched healthy controls plasma fibrinogen and platelet function tests were studied 3-6 months after the MI. At the time of the MI 77% of the patients were smokers but at the time of the investigation 27% of the patients smoked compared to 37% of the controls. Platelet aggregabi1ity was measured in vitro in platelet-rich plasma (PRP) as maximal aggregation to ADP and collagen. The platelet sensitivity to the inhibitory effect of prostacyclin (PG12) was tested by preincubation of PRP with PG12 before inducing aggregation with ADP 5 μM. Plasma levels of beta-thrombog1obuIin (BTG) and platelet factor 4 (PF4) were measured by RIA methods and plasma fibrinogen by heat precipitation. The table presents the results (means ± SE). * is p<0.04, ** is D<0.02 and ns is non significant.Severe emotional stress preceeding the MI occured in 7 patients - these cases had an increased platelet reactivity to ADP. The fibrinogen level was also elevated by smoking and obesity (multivariate analysis). Conclusion: young MI patients have elevated levels ol fibrinogen and reduced platelet sensitivity to PGI2. This might cause an increased thrombotic tendency.

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Physiological Levels of Jak2 V617F Result in Enhanced Megakaryocyte Differentiation, Proplatelet Formation and Platelet Reactivity.

Blood ◽

10.1182/blood.v114.22.226.226 ◽

2009 ◽

Vol 114 (22) ◽

pp. 226-226

Author(s):

Cedric J Ghevaert ◽

Juan Li ◽

Sonia Severin ◽

Jocelyn Auger ◽

Steve Watson ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Platelet Reactivity ◽

Jak2 V617f ◽

Signalling Pathways ◽

Red Cell ◽

Thrombotic Risk ◽

Platelet Production ◽

Bone Marrow Histology

Abstract Abstract 226 The major clinical challenge in treating patients with myeloproliferative diseases (MPD) such as essential thrombocythaemia (ET) and polycythaemia vera (PV) is to prevent thrombotic events. Despite adequate red cell and platelet count control by means of venesection or cytoreductive therapy and the use of anti-platelet agents, patients with ET and PV have a three-fold risk-increase of cardiovascular and cerebral ischaemic events. A V617F mutation in the Janus kinase 2 (Jak2) that causes constitutive activation of Jak2 has been shown to be present in >90% and ∼50% of PV and ET patients, respectively. The effect of this activation on the biology of primary megakaryocytic cells is not known and neither is it clear whether the thrombotic risk conferred by this mutation reflects the increase in red cell/platelet counts, an intrinsic increase in platelet reactivity or both. Studies in humans are hampered by the co-existence of normal and clonal haematopoiesis with significant clonal and phenotypic heterogeneity between patients. Chimaeric mouse models based on retroviral transduction of Jak2 V617F have marked overexpression of the mutant Jak2 and show a PV phenotype with normal platelet counts. Recently, Jak2 V617F transgenic mice models have been published but the transgenes have multiple (and variable) insertions and are therefore subject to position effects with varying degrees of expression of the mutant Jak2 compared to the wild-type allele. We have generated a Cre-inducible mouse model where the human Jak2 V617F gene has been knocked into one allele of the mouse endogenous Jak2 gene. Upon Cre induction, the mice exhibit an ET phenotype (platelet count increased by approximately 30% but with a normal haematocrit) that is stable for over 26 weeks. Bone marrow histology shows classical features of ET with increased numbers of megakaryocytes (MKs) and clusters with no fibrosis. Quantitative RT-PCR shows stable expression of Jak2 V617F in the MKs at a similar level to that of endogenous mouse Jak2 therefore reflecting the physiological situation found in human ET patients. In liquid cultures, bone marrow-derived MKs show increased ploidy in response to suboptimal concentrations of thrombopoietin (TPO) in keeping with the increased number of MKs found in the bone marrow histology. Crucially, in vitro proplatelet formation in mutant MKs was increased two-fold compared to MKs derived from litter-match control animals showing that platelet production may not only relate to the increased number of MKs but to intrinsic differences in MK biology and platelet production. Platelet aggregation studies and P-selectin expression in response to an array of agonists was not significantly different between mutant and controls but in vitro laminar flow assays show increased thrombus formation on collagen. Although Jak2V617F expression was down-regulated at protein level in mature MKs and platelets, analysis of downstream signalling pathways showed alteration of the phosphorylation status of Src kinases. This mouse model therefore provides a unique opportunity to understand the biological mechanism of increased platelet production and thrombotic risk in ET patients as well as unravelling the signalling pathways downstream of the Jak2 V617F in primary cells, which will be crucial in the context of specific therapeutic Jak2 inhibitors currently in clinical development. This work was supported by the British Heart Foundation Disclosures: No relevant conflicts of interest to declare.

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Critical Evaluation of an in Vitro Model for Evaluation of Platelet Reactivity of Biomaterials

MRS Proceedings ◽

10.1557/proc-110-325 ◽

1987 ◽

Vol 110 ◽

Author(s):

Raymond Connolly ◽

Norman Shoenfeld ◽

Karen Ramberg ◽

Allan D. Callow

Keyword(s):

In Vitro Model ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Critical Evaluation ◽

The Body ◽

Test Material ◽

In Vitro Test ◽

Vitro Model

AbstractAn in vitro model for measuring platelet reactivity to a variety of biomaterial candidates for vascular grafts is described. A model consisting of a standard area of test material exposed to freshly labeled In platelets in plasma was evaluated. The platelets were isolated from ACD anticoagulated blood and resuspended in ACD plasma. It has been previously demonstrated that platelets so treated circulate in the body and will deposit on biomaterials exposed to the blood in vivo. The in vitro test consisted of an incubation of the platelets and materials at 37°C for one hour. At the end of the incubation, the platelet rich plasma was removed and the materials washed and removed for gamma counting. Platelet reactivity was normalized as a percentage of the counts on the material to counts in an aliquot of the platelet-plasma incubation media. The maximum uptake of platelets occurred within one hour. Platelets from three species, human, baboon, and dog were tested. Platelet uptake by Dacron and PTFE were in the range of 30–40% and 1–5% respectively. This is in accord with the known reactivity of these two vascular graft materials in vivo.A second series of studies were conducted with physically and pharmacologically inactivated platelets and inert particles. Those studies suggest that the initial results do not represent a biologic event but may reflect the porosity of the materials. This emphasizes the necessity of adequately defining an in vitro model against known in vivo activity.

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Abstract WP269: Increased Sensitivity to recombinant Tissue Plasminogen Activator (rtPA)-induced Lysis in vitro of Thrombi Produced in Human Platelet Rich Plasma containing Dabigatran

Stroke ◽

10.1161/str.44.suppl_1.awp269 ◽

2013 ◽

Vol 44 (suppl_1) ◽

Author(s):

Joanne van Ryn ◽

Monika Kink-Eiband ◽

Davina Fischer ◽

Johanna Schurer ◽

Andreas Clemens

Keyword(s):

Human Platelet ◽

Synergistic Effects ◽

Platelet Rich Plasma ◽

Recombinant Tissue Plasminogen Activator ◽

Thrombin Inhibitor ◽

Similar Degree ◽

Dependent Manner ◽

Experimental Conditions ◽

Human Fibrinogen

Dabigatran is a direct thrombin inhibitor approved for the prevention of stroke in patients with atrial fibrillation. It significantly reduced ischemic and hemorrhagic stroke in these patients vs warfarin. However, in patients treated with dabigtran who developed a stroke, there remain unresolved issues regarding thrombolysis therapy, both in terms of timing of lytic treatment to minimize hemorrhage risk and also lytic dose. It is hypothesized that if the fibrin network in a thrombus formed in patients on dabigatran treatment is altered and is less compact, then it may be more sensitive to lysis at lower doses of rtPA. Thrombi were produced in human platelet rich plasma (PRP) in the presence of low conc. of dabigatran (0, 30, 75 ng/ml). PRP was supplemented with 594-Alexa fluorescent labelled human fibrinogen. Thromboplastin (Thromborel ® ) and 200mM CaCl 2 were added and this was incubated at 37°C for 60 min to allow clot formation. Thrombi were removed from PRP and washed twice in saline. They were then added to human plasma from the same subject containing increasing conc. (0-250 IU/ml) of rtPA (Actilyse ® ) and incubated for 2 hrs under continual stirring conditions at 37°C. Lysis was measured as amount of fluorescence released into plasma from the thrombus over 2hrs. Preformed thrombi added to plasma containing only dabigatran underwent no lysis. Lysis of preformed control thrombi (in the absence of dabigatran) occurred in a conc-dependent manner when incubated with rtPA and maximal lysis was achieved with 300 IU/mL. Thus further experiments were performed with ≤250 IU/ml rtPA to look for potential synergistic effects with dabigatran. Clots preformed in PRP containing either 30 or 75 ng/mL dabigatran were more susceptible to rtPA-induced lysis, with maximal lysis achieved with 100 IU/mL rtPA, and ~ 90% lysis was achieved with 50 IU/mL rtPA. These data provide evidence that thrombi preformed in low concentrations of dabigatran appear to be more susceptible to thrombolysis with rtPA than those preformed in the absence of dabigatran, thus lower concentrations of rtPA achieve a similar degree of thrombolysis under these experimental conditions. This may imply that in patients that suffer a stroke while on dabigatran therapy, lower doses of tPA may also be effective.

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Abstract 606: Antithrombin Nanoparticle Therapy Safely Restores Endothelial Barrier Integrity and Reduces Vessel Hypercoagulability in Atherosclerotic Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.606 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Rohun U Palekar ◽

Andrew P Jallouk ◽

Hua Pan ◽

Samuel A Wickline

Keyword(s):

Endothelial Barrier ◽

Thrombin Inhibitor ◽

Saline Control ◽

Resonance Spectroscopy ◽

Therapeutic Effects ◽

Thrombotic Risk ◽

Barrier Integrity ◽

Vascular Barrier Integrity

Introduction: Thrombin plays a major role in regulating signaling pathways responsible for atherogenesis, hypercoagulability and plaque permeability. Herein, we report the therapeutic effects of perfluorocarbon core nanoparticles (PFC-NP) conjugated to the thrombin inhibitor D-phenylalanyl-L-prolyl-L-chloromethylketone (PPACK-NP) on vascular barrier integrity and hypercoagulability. Methods and Results: ApoE-/- mice were fed a Western diet for 4 months, and received 3 doses/week of saline or 1 ml/kg PPACK-NP for the final month of feeding. Endothelial barrier integrity was assessed by quantifying the ability of atherosclerotic aortas to take up circulating semipermeable PFC-NP (~250 nm diameter). Whole aortas (arch to iliacs) were excised after 2 hour in vivo exposure to PFC-NP and underwent fluorine magnetic resonance spectroscopy ( 19 F-MRS) to quantify plaque-permeating PFC-NP. 19 F-MRS data revealed a significant decrease in plaque permeability to PFC-NP after PPACK-NP treatment compared to saline control (0.081 ± 0.011 μl PFC-NP/g aorta, N = 5 vs. 0.122 ± 0.014 μl PFC-NP/g aorta, N = 8 for PPACK-NP treated vs. saline control, p = 0.027). To assess hypercoagulability, carotid artery injury was induced photochemically to measure the time to complete occlusion as an index of thrombotic risk. Occlusion times were significantly prolonged with PPACK-NP treatment compared to untreated mice (49.8 ± 6.7 min, N = 5 vs. 26.1 ± 4.6 min, N = 9 for PPACK-NP treated vs. saline control, p = 0.019), indicating a decrease in vessel hypercoagulability after therapeutic intervention. Furthermore, PPACK-NP treatment of human aortic endothelial cells in vitro abrogated thrombin-mediated activation of surface PAR-1 receptors as measured by flow cytometry, suggesting a potential dual role for PPACK-NP in the localized modulation of both thrombosis and PAR-1 signaling. Moreover, this sustained therapeutic benefit is obtained without systemic anticoagulation as all clotting parameters and bleeding times are completely normalized within 60 minutes after i.v. injection. Conclusion: Thrombin inhibition with PPACK-NP is effective in restoring vascular barrier integrity and reducing focal thrombotic risk within a single month without incurring bleeding risk.

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