Artlabeling Activity the Phases of Hemostasis and Clot Retraction

Direct inhibition of platelet interactions with fibrin(ogen) past abciximab (Reopro) can restore coronary flow,¹ every bit shown in randomized trials combining abciximab with either primary angioplasty or thrombolysis in acute myocardial infarction.^{2 3 4 5} Notwithstanding, the comprehensive mechanisms remain to be fully understood. In the nowadays study, we sought to demonstrate that this event of abciximab is related to its potential to change platelet-fibrin(ogen) interactions, leading to changes of clot compages and jell physical properties and facilitating pharmacological thrombolysis.

For the commencement step in our experiments, differences between the physical properties of fibrin-rich clots (FRCs) and platelet-rich clots (Cathay) were studied to delineate the mechanical impact of platelets on the fibrin network properties and their consequences for the fibrinolysis rate. The second step in our experiments evaluated the consequences of the inhibition of fibrinogen interactions with platelets on the fibrin network properties past calculation aspirin or abciximab before clotting. In the 3rd set up of experiments, preestablished PRCs were permeated with aspirin or abciximab to evaluate further their power to modulate the interaction between platelets and fibrin. The touch on of these pharmacological changes on the fibrinolysis rate was assessed at each step past either loading recombinant tissue plasminogen activator (rtPA) before the initiation of clotting or by permeating preestablished FRCs and PRCs with rtPA.

Methods

Materials

Abciximab (Reopro) was supplied past Eli Lilly as a 2 mg/mL solution and was dissolved in buffer 1 (0.15 mol/L NaCl and 0.01 mol/Fifty Tris-HCl, pH 7.4). Aspirin (Aspegic) was supplied past Synthelabo (Meudon-La-Forêt) as a 100 mg/mL solution and was dissolved in buffer 1. Murine monoclonal antibodies (anti-CD9 human platelet receptor [Syb]) were provided by Dr Boucheix (CNRS, Villejuif, France). Goat monoclonal anti-mouse antibodies from Amersham Pharmacia were available as 1 mg/mL solution in buffer one. Human thrombin (Enzyme Inquiry Laboratories Inc) was stored at g IU/mL. rtPA was purchased from Boehringer-Ingelheim. FITC and streptavidin-tricolor were from Sigma, and CaCl₂ (0.456 mol/L solution) was from Assistance Publique-Hôpitaux de Paris.

Grooming of Fibrin-Rich and Platelet-Rich Thrombi

Claret was from viii healthy informed volunteers who had no known bleeding disorder and who had not ingested antiplatelet medication for at least 10 days. It was anticoagulated with trisodium citrate (1 vol of 0.13 mol/L citrate for 9 vol blood) and centrifuged at 800g for xv minutes to provide platelet-rich plasma (PRP). Recentrifugation of PRP at 10 0001000 for 15 additional minutes provided platelet-poor plasma (PPP), which was then filtered (0.22-μm filters). PPP was then mixed with the PRP of the same donor to adapt for the final platelet count. Addition of CaCl₂ (20 mmol/L) and thrombin (0.125 IU/mL) to 0.12 mL of either PPP or PRP led to the germination of FRCs and PRCs.

Mechanical Backdrop of Clots

Permeation Experiments

The permeability alphabetize (K _southward) of plasma FRCs and PRCs was measured by the permeation technique.⁶ Briefly, clots were formed in thin glass microchambers (250 μm) and were permeated with buffer 1 at unlike gradients of pressure level (Effigy I, bachelor online at http://atvb.ahajournals.org). The calculated K _{due south} index (in centimeters squared) provides information on the fibrin network compages (shape and size of the pores) and represents the surface of the gel allowing flow.^six The thrombus permeability index equation is every bit follows: K _s=(Q · L · η)/(A · ΔP · t), where Q is the book of liquid (in milliliters) having the viscosity η (ten⁻² poise), flowing through the fibrin gel with length 50 (2.2 cm) and cross section A (0.03 cm²) in a given time t (in seconds) nether a differential pressure ΔP (ranging from 4000 to 10 000 dyne/cm²).

Viscoelastic Experiments

FRCs and PRCs were formed between 2 12-mm-diameter glass coverslips in the torsion pendulum device shown in Effigy II (available online at http://atvb.ahajournals.org) by using the same clotting atmospheric condition every bit for the permeation experiments.^seven Clots had a constant width of i mm. After the initiation of clotting, a momentary impulse was carefully applied to the torsion pendulum arm (air pressure), causing free oscillations of this arm with strains <3%. The frequency of these free oscillations and the rate at which they are damped are functions of the rubberband and pasty backdrop of the clots and are contained of the aamplitude of the initial displacement of the arm.^seven The rigidity index (Grand′, in dynes per centimeter squared), which reflects the viscoelastic properties of the jell, was calculated from the recordings of these oscillations on a chart recorder.

Two different sets of experiment were conducted: (1) FRCs and PRCs that were formed with buffer or antiplatelet agents were candy for Grand _s and G′ measurements. (2) Preformed FRCs and PRCs were advisedly permeated with buffer or antiplatelet agents at a abiding pressure level gradient of 3000 dyne/cm² earlier being processed for M _s and Grand′ measurements. A pocket-size perfusion chamber was added to the torsion pendulum to allow perfusion of the clots at a constant gradient of pressure while the viscoelastic properties were constantly monitored (Figure 2).

Clot Morphological Properties

Confocal Scanning Laser Microscopy Experiments

Clots used for permeation experiments were and then permeated with a 2 mmol/50 FITC solution dissolved in 0.01 mol/L Tris, 0.ane mol/L NaCl, and ane mmol/L EDTA (pH 8) buffer for thirty minutes. The backlog of dye was eliminated past extensive washing with buffer 1.^eight FITC binds to fibrin through nonspecific interactions with the COOH residues. Labeled specimens were scanned with an ACAS 570 interactive laser cytometer (Meridian Instruments) equipped with confocal optics. Twenty-five optical sections were collected at intervals of 1.0 μm in the z-axis and were combined into one prototype, generating a 3D reconstructed prototype of the fibrin network.^six

Platelet Localization Inside the Fibrin Network

Platelets inside the fibrin matrix were specifically localized by using the anti-CD9 antibodies coupled to biotin (25 μg/mL) that were incubated for 5 minutes at 37°C with the PRP before the initiation of clotting. Later clotting occurred, anti-CD9 antibodies were revealed by permeation of the jell with streptavidin-tricolor; fibrin was labeled with FITC as described above. After washing, platelet-rich areas containing fibrin-FITC and tricolor platelets appeared yellow; fibrin-FITC areas without platelets appeared dark-green.

Prototype Analysis

Quantitative assay of the average area of platelet aggregates (S.ag, in micrometers squared) was performed with the Visilog software (version v.01, Noesis). Because aggregates and the surrounding fibrin agglomerated within the reconstructed images from confocal scanning laser microscopy, an optical algorithm based on "the watershed line" transformation technique was required to separate them before being measured.^{ix 10}

Lysis Experiments

Fibrinolysis was assessed by monitoring of the viscoelastic index K′ every 4 minutes, and the fibrinolysis speed corresponded to the time taken for G′ to decrease to 50% of its maximal value.^xi Clots were formed in the exact same conditions equally for viscoelastic measurements, and 2 dissimilar assays were developed: (1) In the static lysis assay, rtPA was added with buffer or with antiplatelet agents before the initiation of clotting. rtPA concentration (1 or ii nmol/Fifty) was adjusted so that lysis started later the beginning 10 minutes of initiation of clotting.¹¹ (2) In the dynamic lysis analysis, preformed PRC was carefully permeated with rtPA and buffer or antiplatelet agents at a terminal concentration of 0.2 μmol/L, which is the in vivo predicted concentration from computer simulation of thrombolysis.¹² The unlike rtPA concentrations used in these two assays reverberate the divergence of their design: in the static assay, rtPA is already in the fibrin fibers, whereas in the dynamic lysis assay, rtPA needs to be delivered to the fibrin network under pressure-driven permeation.¹³

Comparison of the Efficacy of Aspirin and Abciximab in Remodeling the Anatomy of the Jell

To simulate in vitro the dynamic changes in the structure and the lysis speed of an occlusive coronary thrombosis after it is exposed to antiplatelet agents in vivo, occlusive and fully hydrated FRCs and PRCs were used to evaluate the potential of aspirin and abciximab to either prevent fibrinogen interactions with platelets or to remove platelets from fibrin. Aspirin and abciximab were either added before clotting initiation or advisedly infused through preformed clots. Permeation started v and 40 minutes after clotting initiation for a 20-minute time menses at a constant gradient of pressure of 3000 dyne/cm^two. Fibrinolysis was assessed by loading rtPA in combination with either aspirin or abciximab before the initiation of clotting (static lysis assay) or by permeating rtPA (0.two μmol/L) in combination with aspirin or abciximab (dynamic lysis assay). Control clots were formed and/or permeated with buffer 1. G′ and K _south were measured earlier, during, and after permeation. Clots used for permeation experiments (without rtPA) were and then processed for microscopy.

Aspirin concentrations (50 and 100 μg/mL) correspond to an intravenous administration of 500 and one thousand mg aspirin, which completely inhibits platelet cyclooxygenase activity.^fourteen Abciximab concentrations (0.034 and 0.068 μmol/L) correspond to an intravenous bolus administration of 0.15 and 0.30 mg/kg, which produces near-binding saturation of platelet surface glycoprotein (GP) IIb/IIIa receptors and inhibits thrombosis in vivo.¹⁵ This was farther ascertained by the use of the rapid platelet functional assay (Accumetrics), which confirmed that 99% of the platelet GP IIb/IIIa receptors were occupied with both abciximab concentrations, whereas 95% and 100% of the platelet receptors remained free with aspirin at fifty and 100 μg/mL, respectively.^sixteen These abciximab concentrations were also shown to inhibit 75% and 100% of the mechanical effect of platelets on fibrin, respectively.¹⁷

All experiments were conducted with a range of platelets of 75 000 to 150 000/μL. A dose-related effect of platelets on mechanical backdrop and on fibrinolysis speed was found. Results with a final platelet count of 75 000/μL are presented here, particularly because measurements of permeability abiding K _s and morphological index Southward.ag were more accurate than with 150 000 platelets per microliter.

Statistical Analysis

Statistical assay was performed with StatView software. Continuous variables were expressed every bit mean±SEM, and grouping differences were adamant by ANOVA. The Student t test for paired samples was used to take into account the heterogeneity amid the plasma of the viii donors. A value of P<0.05 was considered to indicate statistical significance.

Results

Comparison Between FRC and PRC Properties

Intra-assay and interindividual variabilities in permeability and viscoelastic experiments were similar to those previously reported for plasma FRCs.⁸ The coefficients of variation for K _southward and G′ were 12.v% and 9.2%, respectively, for a double measurement on a single sample (intra-assay variability, northward=8). Measurement of Thousand _s was performed in different chambers. The coefficient of variation of K _s was slightly college when platelets were added earlier clotting (16.five%, n=8).

Platelets increased the whole-clot permeability (Grand _{due south}) by 3.v-fold and the whole-jell rigidity (G′) by 3-fold (Table). These platelet-dependent modifications of the physical properties of the fibrin network were ascertained further by the opposite correlation (R) betwixt K _s and Chiliad′ in PRCs (R=0.55, P=0.02) compared with FRCs (R=−0.64, P=0.0061; Figure ane). It is likely that platelets within the PRC simultaneously strengthen and distort the fibrin network, resulting in more than space inside the pores delimited past fibrin fibers. This was confirmed by confocal microscopic analysis of the same clots. A typical FRC appeared as a homogeneous structure made up of direct rodlike elements corresponding to branching and crossing fibrin fibers organized in a 3D network (Figure 2A). The round white elements correspond to branching fibers perpendicular to the aeroplane of the scanning expanse. PRCs were rather inhomogeneous and were characterized by aggregates of platelets, for which the average surface area was 5562±521 μm² (n=8, Figure 2B). Fibrin fibers located within and at the edge of these aggregates were bent every bit if they were stressed and trapped. The double fluorescence labeling experiment showed that these aggregates corresponded to the colocalization of platelets and fibrin (Effigy 2C). Hence, superposition of fibrin-FITC and platelet-tricolor within the aggregates appeared every bit yellow areas, and FITC-fibrin alone appeared as green areas surrounding the platelet-fibrin amass (Figure 2C).

Platelet-mediated fibrin retraction led to a ii.6-fold increase in the lysis fourth dimension of PRCs compared with FRCs formed in the same conditions and with use of the static lysis assay (ane nmol/50 rtPA, Effigy 3a). Raising the rtPA concentration up to 2 nmol/L lowered the average lysis time of PRCs by 32.viii±20% (north=16, P<0.0001), only information technology still remained significantly higher than that of FRCs with i nmol/L of rtPA (Effigy 3a).

Event of Adding Antiplatelet Agents Earlier Clotting

Unlike FRCs, mechanical properties of PRCs were greatly affected by the addition of abciximab or aspirin earlier the initiation of clotting (Table). In all treated groups, a pregnant subtract in Grand _{due south} and G′ was establish. Unlike aspirin, abciximab displayed a dose-dependent efficacy in reducing G′ simply non G _s. Unlike aspirin, abciximab (0.068 μmol/50) reversed the relation betwixt K _{due south} and G′, but in a nonsignificant mode (R=−0.28, P=0.28; Figure one). This relation was intermediate betwixt the positive one of PRCs (R=0.55, P=0.02) and the negative one of FRCs (R=−0.64, P=0.0061). This indicates that despite a well-nigh consummate inhibition of fibrinogen-platelet interactions, as shown past the measurement of GP IIb/IIIa receptor occupancy with the Accumetrics device, abciximab failed to restore a platelet-costless–like fibrin structure. The significantly higher values of G′ and Grand _s of PRCs formed with 0.068 μmol/L of abciximab compared with FRCs further support this assumption. It seems probable that interactions betwixt platelets and fibrin differ from interactions between platelets and fibrinogen.

These mechanical changes are correlated with morphological changes. Hence, adding abciximab or aspirin before Communist china formation significantly decreased the average area of platelet-fibrin aggregates (Southward.ag), with a tendency for a dose-dependent effect (Table and Figure second and 2E). Yet, the only significant reduction betwixt treated groups was plant between 50 μg/mL of aspirin and 0.068 μmol/50 of abciximab. These morphological changes resulted in a more homogeneous fibrin architecture with more fibrin exposed within the fibrin network, which is consistent with the concomitant reduction of both Grand _south and Chiliad′ (Table).

Adding abciximab (0.034 μmol/50) and rtPA (one nmol/L) before the initiation of clotting reduced the boilerplate lysis time of PRCs by 36.7±eleven% (P<0.001) compared with PRCs formed without abciximab. Add-on of abciximab had the same effect as a ii-fold increment in the rtPA concentration (20.2±2.seven and 20.75±3.95 minutes, respectively; P=0.77; Effigy 3a). Although nonsignificant, there was a trend for a dose-related efficacy of the profibrinolytic effect of abciximab. The lysis rate of PRCs formed with 0.068 μmol/50 abciximab still remained slower than the lysis charge per unit of FRCs at a similar concentration of rtPA (P=0.019); this was consistent with the remaining departure in the physical properties between these 2 types of clots (Figure 3a). Aspirin at 50 μg/mL increased the lysis rate of PRCs by 21±8% (due north=eight, P<0.01), simply unlike abciximab, there was no trend for a dose-related event (not shown).

Effects of Permeating Preestablished PRCs With Antiplatelet Agents

Permeation of preformed FRCs with aspirin or abciximab did non affect the physical properties of fibrin. Unlike buffer or aspirin, permeation of PRCs at a abiding force per unit area slope with 0.068 μmol/L abciximab significantly reduced M _southward and K′, whereas S.ag was decreased in a nonsignificant manner (Tabular array and Figure 2F). Interestingly, these changes were found to be time-related. Early on perfusion, starting within the 10 first minutes of clotting, was effective, whereas late perfusion, starting 40 minutes subsequently the initiation of clotting, had no significant effect (Tabular array).

Permeation of preformed PRCs with rtPA (200 nmol/Fifty) and aspirin (100 μg/mL) had an effect on the lysis time like to that obtained past permeation with rtPA alone. Unlike aspirin, permeation of PRCs with abciximab (0.068 μmol/L) and rtPA (200 nmol/Fifty) produced a 27% increase of the lysis speed (P<0.001, Effigy 3b). This accelerating result of abciximab was not dose-related but time-related. As for mechanical properties, it was found with early permeation (starting within the first ten minutes of initiation of clotting) but non with late permeation (starting after 40 minutes).

Give-and-take

The present work was designed to evaluate the effect of platelets and platelet GP IIb/IIIa antagonists on fibrin physical properties and the fibrinolysis charge per unit of native fully hydrated PRCs. Information technology was hypothesized that inhibition of the interactions of platelet-membrane receptor GP IIb/IIIa with either fibrinogen or fibrin could modify the architecture of PRCs, leading to an acceleration of its dissolution. The near important finding of this in vitro report is that unlike aspirin, early permeation of preformed PRCs with abciximab can disaggregate these clots and facilitate fibrinolysis. These results provide new information on the mechanisms of abciximab to achieve total reperfusion of initially occluded coronary arteries.

Touch on of Calculation Platelets and Antiplatelet Agents Earlier Clotting on Physical Properties and Fibrinolysis Charge per unit of PRCs

Our results provide for the offset time straight correlations between mechanical and morphological properties of fully hydrated clots. Compared with FRCs, PRCs are stiffer, more porous (increment of G′ and Chiliad _s, respectively), and more heterogeneous, considering of the presence of platelet-fibrin aggregate-similar structures. These platelet-related changes of the fibrin physical properties account in part for the fibrinolysis resistance of PRCs. Calculation antiplatelet agents before clotting prevented platelet-related mechanical (reduction of Yard′ and K _s) and morphological changes (reduction of S.ag) of the fibrin network, leading to a significant increase of the fibrinolysis charge per unit. Unexpectedly, decreased permeability was plant to be associated with increased lytic speed in the static lysis assay. This is because fibrin, which was trapped into the platelet-fibrin aggregates, has been reexposed in the surrounding fibrin network every bit a event of the inhibition of the interaction between fibrinogen and platelets by abciximab. Consequently, more fibrin is accessible to rtPA, which is loaded earlier the initiation of clotting, leading to a faster fibrinolysis, but the overall permeability of the clot is decreased.

The superiority of abciximab over aspirin in this setting was plant to be straight related to a better inhibition of the fibrinogen-platelet interactions. Although aspirin provided a complete inhibition of the platelet cyclooxygenase activity, nearly all of the GP IIb/IIIa receptors remained functional after platelet activation with thrombin. The dose-related effect of abciximab that is reported in the present study farther confirms previous in vivo pharmacodynamic studies showing a dose response of abciximab bounden to platelet GP IIb/IIIa receptors.^xv Interestingly, although simply few GP IIb/IIIa receptors remained unoccupied with the highest dose of abciximab (0.068 μmol/L), a significant difference betwixt the physical backdrop and the fibrinolysis rate of PRCs and FRCs was found. This finding is consistent with recent studies using recombinant fibrinogens showing that platelet interactions with fibrin may involve receptors other than GP IIb/IIIa.^{xviii 19} Furthermore, the nowadays written report corroborates previous data showing that at 0.034 to 0.068 μmol/L abciximab, platelet-mediated fibrin retraction is not completely abolished, although there is a consummate inhibition of platelet assemblage at ≥0.034 μmol/L abciximab.²⁰

Bear upon of Permeation of Antiplatelet Agents and/or rtPA on Physical Properties and Fibrinolysis Charge per unit of Preformed PRCs

To consider rheological, send, and enzymatic events, preformed PRCs were permeated at a constant gradient of pressure with antiplatelet agents and/or rtPA. Every bit expected, PRCs were constitute more resistant to fibrinolysis than were FRCs. These findings are consequent with previous studies showing a decreased binding of tPA in PRCs as a event of platelet retraction.^{21 22} Dissimilar aspirin, permeation of PRCs with abciximab significantly decreased 1000 _southward and One thousand′, suggesting that fibrin is removed from preexisting aggregates and reexposed into the surrounding fibrin network, every bit confirmed by confocal microscopy (subtract of S.ag). Interestingly, these abciximab-related changes of the fibrin network construction were found to exist fourth dimension-related and were observed only with early permeation (<ten minutes) and led to a faster fibrinolysis. Therefore, information technology is likely that the dissociating chapters of abciximab depends on the age of the PRCs and that the structure of the PRCs changes over time. This was ascertained past the continuous increase of G′ up to 40 minutes later the initiation of clotting and relates to the development of the platelet-contractile strength that slowly retracts the fibrin network.²³ Therefore, nosotros assume that within the offset 10 minutes of the initiation of clotting, the dissociating capacity of abciximab is strong enough to reverse the interactions between fibrin and activated platelets,²⁴ whereas after 40 minutes, tight contacts between fibrin and platelets may prohibit the large molecule of abciximab (molecular weight of 47 000) to access the GP IIb/IIIa receptors. The fact that the interactions of fibrin with platelets may involve receptors other than GP IIb/IIIa, equally shown above, is an important boosted explanation for the limited efficacy of permeation of abciximab in disaggregating PRCs.

Relations of Our Experimental Blueprint to Clinical Realities

Our information chronicle to clinical realities in different ways. Coronary thrombosis involves simultaneous deposition and lysis of ruddy (fibrin-rich) and white (platelet-rich) clot components, causing intermittent vessel apoplexy. Although this dynamic aspect of coronary thrombosis could not be directly assessed in our experimental design, our information clearly emphasize that abciximab could affect simultaneously preformed PRCs (disaggregating potential) and clot extension by the prevention of platelet-related fibrin remodeling. Moreover, it is probable that the potential of abciximab to reduce the generation of thrombin may accentuate the limitation of the clot extension in vivo, although this has not been directly demonstrated in our work.²⁵ Hence, fibrin expression from platelets plays a minor role in our experimental system because fibrin formation is mainly related to the activation of fibrinogen molecules by the exogenous thrombin addition. Thrombin that is generated as a consequence of platelet activation has a unlike kinetic profile and ordinarily appears later on the activation of nearly all the fibrinogen molecules.

The fourth dimension-related efficacy of abciximab in disaggregating preformed PRCs is consistent with the temporal relationship detected betwixt the age of the thrombus and the efficacy of abciximab in reperfusing the occluded coronary avenue in vivo.²⁶ These in vitro information further back up contempo results of randomized trials showing that abciximab with reduced-dose fibrinolytic agents enhances fibrin and platelet lysis, resulting in rapid and complete reperfusion (inside hour) in a loftier proportion of patients with myocardial infarction (near lxx%).^{4 5} Moreover, our findings are consistent with those of the same trials showing that adding low-dose reteplase to abciximab provides slightly superior rates of culprit artery patency compared with those with standard-dose reteplase alone (run into Effigy 3).

Study Limitations

In terms of clot mechanics, final platelet count is plainly a critical parameter and therefore a limitation of our experimental design. We institute a correlation betwixt clot strength and platelet count with a dose-related enhancement of G′, every bit described before: G′=420+p^0.76 (where p is platelet concentration).²⁷ In parallel, the whole-jell permeability, K _s, was institute to increase linearly with platelet concentration. However, accurate measurements of K _s were impaired at >100 000 platelets per microliter because retraction led to the germination of leaks. For the aforementioned reasons, morphological analyses were not possible. Fibrinolysis resistance using the static lysis analysis was institute to be positively correlated with the platelet concentration, and the result of abciximab on physical backdrop and the fibrinolysis charge per unit was of a similar magnitude over the whole range of platelet concentration.

The question arises as to whether our experimental weather condition are relevant to an in vivo scenario, especially regarding the retraction phenomena. Blood-red blood cells are an important component of the thrombus, primarily considering their incompressibility limits clot retraction to l%.¹³ In our system, scarlet blood cells were discarded because the organization did not permit optical microscopy to be performed; however, the high surface area of contact of the clots within the microchamber considerably limited the retraction phenomena. Therefore, we presume that a terminal platelet count of 75 000 per microliter may take a like impact on the fibrin network as a platelet concentration of 150 000 per microliter in the presence of red blood cells.

Although the nowadays written report emphasizes the part of PRC architecture equally an important determinant of the fibrinolysis rate, other factors are of major importance. Plasminogen activator inhibitor blazon 1, which has been shown to be a major determinant of the failure of thrombolytic therapy in vitro and in vivo, may take an of import part in our experimental setting.^{28 29 xxx} It has been detected but in platelet-fibrin aggregates and with a higher intensity in PRCs formed with buffer compared with abciximab. Further quantitative analyses are needed to evaluate the bear on of plasminogen activator inhibitor type 1 in our setting. A structural and dynamic approach to the early stage of People's republic of china germination and fibrinolysis is also an important upshot and is likewise needed to evaluate our findings and to extend previous work on the concept of a heterogeneous architecture of occlusive PRCs with aggregates that resist fibrinolysis.^{31 32 33}

In determination, inhibition of platelet-mediated fibrin remodeling and removal of fibrin from preexisting aggregates contribute to the disaggregating potential of abciximab and are potential mechanisms for its additional benefit over aspirin in the setting of thrombolysis in myocardial infarction.

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Figure 1. Correlation between Chiliad′ (rigidity alphabetize) and M _s (permeability index) in FRCs and PRCs, respectively. FRCs are characterized past a negative correlation betwixt M′ and K _s (open triangle, r=−0.64, P=0.0061). Platelet-mediated fibrin remodeling reverses this correlation in PRC (solid triangle, r=0.55, P=0.02). Inhibition of platelet-mediated fibrin remodeling by the improver of abciximab (Abc, 0.068 μmol/L) before the initiation of clotting lowers G′ and K _s, which become negatively related (solid diamond, r=−0.28, P=0.28).

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Figure 2. Architecture of fully hydrated clots visualized past confocal microscopy. A, FRCs are made up of fibers (arrow) organized in a homogeneous 3D network delimiting pores (arrowhead). B, In PRCs, platelets are organized in aggregates detected by the watershed line segmentation method and distort the architecture of the surrounding fibrin network. C, The double fluorescent labeling allows specific localization of fibrin and platelets. FITC-fibrin is green, and tricolor platelets are red. Yellow areas, which stand for to a colocalization of fibrin and platelets, are located within the aggregate. D and E, Addition of aspirin (100 μg/mL, D) or abciximab (0.068 μmol/Fifty, E) before PRC formation reduces platelet-mediated fibrin remodeling. F, Permeation of preestablished PRCs with abciximab (0.068 μmol/Fifty) also reduces the platelet-mediated fibrin remodeling. Each micrograph is a 2D projection of an initial book of 100×100×25 μm³. Bar=10 μm (final platelet count 75 000/μL).

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Figure 3. Affect of aspirin and Abc on the lysis speed of PRCs. Aspirin (Asa, in μg/mL), Abc (in μmol/L), and rtPA (t-Pa, in nmol/L) were either added earlier the initiation of clotting (a) or infused through preestablished Cathay within the first x minutes of clotting initiation (b). Control clots were formed without antiplatelet agents. Fibrinolysis was assessed past the evaluation of the lysis time (in minutes) that corresponded to the time taken for the rigidity index G′ to subtract to 50% of its maximal value. Addition of Asa and Abc before the initiation of clotting significantly increased the lysis rate. Addition of Abc (0.034 μmol/L) had an effect on lysis acceleration similar to that with a 2-fold increase in rtPA concentration. Early on perfusion of Abc, but not Asa, through preestablished PRCs significantly decreased the lysis fourth dimension past 27% (P<0.01).

Table 1. Effect of Platelet and Antiplatelet Agents on Fibrin Physical Properties of FRCs and PRCs

	1000 south, 10−8 cm2	G′, dyne/cm2	S.ag, μmii
Control FRCs	eighteen ±v	361±11	ND
Command PRCs	84 ±5i	1032±171	5561±521
PRCs formed with Asa l	45±32 (46%)	576 ±102 3 (44%)	4973±3122
PRCs formed with Asa 100	42±44 (l%)	607 ±114 5 (41%)	3681±584 (32%)
PRCs formed with Abc 0.034	38±3 (55%)	484±seven (53%)	3293±435 (40%)
PRCs formed with Abc 0.068	25±iii (70%)	349 ±vii (66%)	2114±207 (61%)
Early perfusion of PRCs with Asa 100	75±14 (six%)	796±133 (seven%)	5342±331 (iv%)
Early perfusion of PRCs with Abc 0.068	50±v (37%)	557 ±82 (35%)	4251±501 (25%)
Late perfusion of PRCs with Abc 0.068	70±12 (6%)	832±151 (7%)	5443 ±421 (4%)

Presented in part at the 71st Scientific Sessions of the American Heart Association, Dallas, Tex, Nov 8–xi, 1998.

This written report was supported by a grant from the Association de Cardiologie Ile-de-France, Paris, France. We wish to thank J.W. Weisel, PhD, from the Department of Cell and Developmental Biology, University of Pennsylvania (Philadelpha, Pa), for the fruitful discussion and his proficient review of the manuscript.

Footnotes

Correspondence to J.P. Collet, MD, PhD, Section of Cardiology, Middle Hospitalier Universitaire Pitié-Salpêtrière, 47, Boulevard de l'Hôpital, 75013 Paris, France. E-postal service [e-mail protected]

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Source: https://www.ahajournals.org/doi/10.1161/01.atv.21.1.142

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