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PubMed1997

Nitric oxide inhalation inhibits platelet aggregation and platelet-mediated pulmonary thrombosis in rats.

Endothelium-derived nitric oxide (NO) inhibits in vitro platelet aggregation via a cGMP-dependent mechanism. The effect of inhaled NO on platelet-mediated pulmonary thrombosis following intravenous thrombotic challenge with collagen was examined in rats and compared with the effect of G4120, a cyclic Arg-Gly-Asp-containing synthetic pentapeptide that binds to the platelet glycoprotein IIb/IIIa receptor. Intraplatelet cGMP dose-dependently increased from 39 +/- 6 fmol/10(8) platelets in control to 46 +/- 6, 68 +/- 13, and 81 +/- 13 fmol/10(8) platelets after inhalation with 20, 40, and 80 ppm NO, respectively (P < .05 for 40 and 80 ppm). Ex vivo platelet aggregation of platelet-rich plasma induced by 1 microgram/mL collagen was reduced from 75 +/- 4% in control rats to 22 +/- 10% and 20 +/- 7% in rats ventilated with 40 and 80 ppm NO, respectively, and to 30 +/- 9% in G4120-treated rats (each P < .05 versus control). Circulating platelet counts 3 minutes after collagen injection were significantly higher in the inhaled NO and G4120 groups compared with control rats (250,000 +/- 18,000 and 223,000 +/- 10,000/microL versus 160,000 +/- 18,000/microL, each P < .05). The rise in pulmonary arterial pressure after collagen injection was significantly reduced in NO- and G4120-treated rats (26 +/- 1 and 27 +/- 1 versus 32 +/- 1 mm Hg in control rats, each P < .05). The number of pulmonary resistance vessels containing platelet thrombi was significantly smaller after inhaled NO and G4120 treatment compared with control (56 +/- 3% and 50 +/- 3% versus 68 +/- 3%, respectively; P < .05). Thus, NO inhalation reduces in vivo activation of circulating platelets and platelet-rich thrombosis in thromboembolic pulmonary hypertension. Inhalation of NO may be useful in cardiovascular diseases associated with platelet activation.

Administration, Inhalation↗
PubMed1993

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

Amino Acid Sequence↗
PubMed2003

The effect of plasma depletion of platelet concentrates on platelet aggregation and phosphatidylserine expression.

Plasma-reduced platelet concentrates are commonly administered to prevent febrile transfusion reactions and to avoid fluid overload in neonates. Because little is known about the influence of centrifugation and resuspension on functional aspects of platelets, we examined the effects of plasma-reduction on platelet aggregation and platelet-dependent thrombin generation. Our results show that plasma reduction and resuspension of the platelet pellet in saline or plasma results in a significant reduction in platelet aggregation to a combination of the platelet agonists adenosine diphosphate and epinephrine (p < 0.001). In contrast, when a combination of the more potent agonists collagen and thrombin was used, platelet aggregation was maintained. Likewise, no decline was observed in platelet-dependent thrombin generation as measured by the functional prothrombinase assay or Annexin V binding. We conclude that centrifugation and resuspension of platelets to render the concentrate plasma-free, as a routine procedure in blood banking, variably affects in vitro platelet aggregability but does not significantly affect platelet-dependent thrombin generation.

Annexin A5↗
PubMed2006

Disabled-2 is a novel alphaIIb-integrin-binding protein that negatively regulates platelet-fibrinogen interactions and platelet aggregation.

Platelet aggregation plays a pivotal role in the haemostatic process and is involved in the pathological counterpart of arterial thrombosis. We have shown that the adapter protein disabled-2 (DAB2) is expressed abundantly in platelets. In this study, DAB2 was found to distribute in the platelet alpha-granules and was released from the granular compartment upon platelet activation. The secreted DAB2 binds to the extracellular region of alphaIIbbeta3 integrin on the platelet surface through the phosphotyrosine-binding domain. The DAB2-platelet interactions result in the inhibition of agonist-induced platelet aggregation with the exception of thrombin, a DAB2 protease that renders DAB2 inactive. Biochemical and mutational analysis revealed that the DAB2 cell-adhesion Arg-Gly-Asp (RGD) motif (amino acid residues 64-66) and the alphaIIb-integrin-fibrinogen-binding region (amino acid residues 171-464) are important for the DAB2-platelet interactions. Such interactions compete for the binding of alphaIIb integrin with fibrinogen and provide a mechanism for DAB2 to inhibit platelet aggregation. Accordingly, the synthetic RGD-motif-containing DAB2 peptide PDARGDKM also elicited anti-platelet aggregation activity. These findings demonstrate for the first time that DAB2 is an alphaIIb-integrin-binding protein that plays a novel role in the control of platelet-fibrinogen interactions and platelet aggregation.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton.

This study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100-insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension-activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent-resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.

Adult↗
PubMed1992

von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin.

We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin-induced platelet activation is a potential mechanism for regulating platelet adhesivity.

Antibodies↗
PubMed1992

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.

Adenosine Diphosphate↗
PubMed1988

Temporary inhibition of platelet function with iloprost (ZK36374) preserves canine platelets during extracorporeal membrane oxygenation.

Contact between blood and artificial surfaces results in extensive quantitative and qualitative alterations in platelet function. We evaluated the efficacy of a brief infusion of iloprost (ZK36374), a stable analog of prostacyclin, in preventing these platelet changes during extracorporeal membrane oxygenation. Twelve nonsplenectomized male mongrel dogs (23 to 30 kg) were randomized to treatment (n = 6) and control (n = 6) groups. The treatment animals received an infusion of iloprost at a rate of 150 ng/kg/min with the infusion being terminated 30 minutes after the initiation of extracorporeal membrane oxygenation, despite the fact that all animals were maintained on extracorporeal membrane oxygenation for 3 hours. In the control group, platelet counts dropped to 54% +/- 8.9% (mean +/- standard error of the mean) of initial levels at 30 minutes of extracorporeal membrane oxygenation and gradually rose to 87.2% +/- 6.7% at 3 hours. In contrast, the platelet counts of the iloprost-treated dogs remained stable throughout extracorporeal membrane oxygenation at 98.3% +/- 4.2% of initial counts. Platelet reactivity toward adenosine diphosphate revealed a significant and permanent loss of platelet function in the control group (37.0% +/- 2.1% inhibition). In contrast, the iloprost group demonstrated significant inhibition of platelet reactivity (79.2% +/- 8.3%) during the iloprost infusion but a return to normal function (4.2% +/- 6.7% inhibition) after cessation of drug infusion which persisted throughout extracorporeal membrane oxygenation. Plasma levels of the platelet-specific protein thrombospondin rose progressively from 918 +/- 89 ng/ml to 1465 +/- 239 ng/ml (delta 548 +/- 179 ng/ml) at 30 minutes of extracorporeal membrane oxygenation, which indicates extensive release of platelet granule contents (p less than 0.05). In contrast, plasma thrombospondin levels in the iloprost group demonstrated no additional rise after cessation of the iloprost infusion. In conclusion, iloprost effectively preserves platelet number and function during extracorporeal circulation. The fact that its salutary effects outlast its presence in plasma suggests that prevention of initial platelet-synthetic surface interactions permits the appearance of reduced surface affinity for platelets and, thus, reduced synthetic surface thrombogenicity.

Animals↗
PubMed1987

Monoclonal antibodies specific for human platelet membrane glycoproteins bind to monocytes by focal absorption of platelet membrane fragments: an ultrastructural immunogold study.

The membrane labeling of monocytes by monoclonal antibodies directed against platelet glycoproteins Ib (AN51), IIb (Tab), IIIa (C17), IIb-IIIa complex (J15) and to antigens common to platelets and monocytes (anti-monocyte platelet antigen and FA6 152) has been investigated by an ultrastructural immunogold method. Only with FA6 152, which identifies a structure shared by erythroblasts, platelets, and monocytes, was labeling obtained on membranes of both platelets and monocytes from normal blood. With all the other monoclonal antibodies, platelets were highly labeled but monocytes lacked quantitatively significant label; however, focal microparticles which exhibited gold particles were adherent to the membrane of monocytes. This localized labeling, interpreted as resulting from the fragmentation of platelet membranes during monocyte isolation with adhesion of the fragments to monocyte surfaces, was verified by two approaches. First, double staining with C17 visualized by an anti-IgG coupled to 40 nm gold particles and MO2 recognizing exclusively a surface monocyte antigen, as visualized by an anti-IgG coupled to 15 nm gold particles, was performed. The absence of colocalization of large and small gold particles either on monocytes or on microparticles confirmed the exclusive cell origin. Second, when analyzed by quantitative x-ray analysis, monocyte associated gold following C17 treatment was restricted to platelet pseudopods and fragments on whole mount spread cells. Finally, when monocytes were spread immediately after blood collection in the absence of sedimentation and centrifugation to prevent platelet activation, platelet rosetting was avoided and the number of microparticles markedly decreased. Thus, the attachment to monocyte membranes of microparticles originating from platelets may be confused with true labeling of monocytes by antibodies to platelet glycoproteins if analysis is limited to immunofluorescence.

Adsorption↗
PubMed1982

Specific binding of phospholipid platelet-activating factor by human platelets.

The binding of the phospholipid platelet-activating factor 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) to washed human platelets was more than 80% complete within 2 min, which coincided with the time of initiation of platelet aggregation by AGEPC. Scatchard plot analysis of the binding of [3H]AGEPC to platelets without and with an excess of unlabeled AGEPC revealed two distinct types of binding sites. One platelet site for AGEPC exhibited a high affinity (KD = 37 +/- 13 nM, mean +/- SD), was saturable, and had a low maximal capacity of 1399 +/- 498 (mean +/- SD) molecules of AGEPC/platelet. The other platelet site demonstrated a nearly infinite binding capacity, consistent with nonreceptor uptake of AGEPC into cellular structures. The specificity of the high-affinity binding site for AGEPC was assessed by comparing the capacity of several analogues of AGEPC to inhibit the binding of [3H]AGEPC to platelets and to induce platelet aggregation. An ether linkage in position 1, a short-chain fatty acid in position 2, and a choline moiety in the polar head group proved to be critical both for the binding of [3H]AGEPC to platelets and for the initiation of platelet aggregation. Exposure of platelets to AGEPC for 5 min at 37 degrees C functionally deactivated the exposed platelets to subsequent stimulation by AGEPC, as assessed by diminished aggregation, and concomitantly reduced the specific binding of [3H]AGEPC. Evaluation of the time course of the events of deactivation revealed the loss of an aggregation response to AGEPC after 90 sec at 37 degrees C, despite the retention of up to 50% of the specific binding sites for AGEPC.

Blood Platelets↗
PubMed1994

The platelet adhesion capacity to subendothelial matrix and collagen in a flow model during storage of platelet concentrates for 7 days.

The influence of storage of platelet concentrates (PC) on the adhesion capacity of platelets was studied. Twenty-four PC, 12 prepared by the buffy coat (BC) method and 12 by the platelet-rich plasma (PRP) method, were stored for 7 days at room temperature. On days 1, 3 and 7 of storage, the platelet adhesion capacity to subendothelial matrix (SEM) and collagen was studied in a rectangular perfusion system under flow conditions in conjunction with the platelet aggregation capacity after stimulation and the adenine nucleotide content. The platelet adhesion capacity to collagen was constant until day 3 of storage and decreased to about 80% of the starting value on day 7 of storage. The adhesion capacity to SEM, however, had already decreased on day 3 to about 75% of the value of day 1 and was even more decreased on day 7 to about 45% of the starting value. On day 1, platelets prepared by the BC method displayed a higher adhesion capacity to collagen and a higher aggregation capacity after stimulation by collagen alone or in combination with ADP, compared to platelets prepared by the PRP method. No other significant differences in adhesion or aggregation capacity were observed between the PC prepared by the two different methods. Both platelet adhesion and aggregation response decreased during storage, as did the total adenine nucleotide content. This study shows that platelet function, as measured by the aggregation and adhesion capacity, of platelets prepared by the PRP method is more severely impaired during the first 3 days of storage as compared to the function of platelets prepared by the BC method.

Blood Platelets↗
PubMed1996

Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura.

Canine idiopathic thrombocytopenic purpura (ITP) is clinically analogous to chronic ITP in human beings. The objective of this study was to investigate the pathogenesis of canine ITP by determining whether immunoglobulins bound to the surface of platelets from dogs with ITP (platelet-bound immunoglobulins) were directed against host platelet antigen and whether platelet glycoproteins (GP) IIb and IIIa were target antigens in dogs with ITP. Thirty-two dogs with ITP were studied. Increased platelet-bound immunoglobulin concentrations were detected in 30 cases (94%), and increased concentrations of serum platelet-bindable immunoglobulins were detected in 11 cases (34%). Immunoglobulins eluted from the surface of platelets from dogs with ITP bound to homologous normal canine platelets in 11 of 19 cases (58%). Immunoglobulins against platelet membrane GP IIb and/or IIIa were detected in serum from four of 17 affected dogs. This study provides evidence that immunoglobulins bound to surface of platelets from some dogs with ITP are directed against host platelet antigens and that platelet target antigens are, in some cases, GP IIb and IIIa. This supports the hypothesis that canine ITP is an autoimmune disease, similar to the pathogenesis of chronic ITP in human beings.

Animals↗
PubMed1997

Platelet prothrombinase activity and intracellular calcium responses in patients with storage pool deficiency, glycoprotein IIb-IIIa deficiency, or impaired platelet coagulant activity--a comparison with Scott syndrome.

The procoagulant activity of platelets induced by collagen, thrombin, and collagen plus thrombin, measured as their capacity to promote the conversion of prothrombin to thrombin in the presence of factors Va and Xa, was studied in patients with alpha, alpha delta, and delta storage pool deficiency (SPD), thrombasthenia, and in two new patients with isolated defects in platelet coagulant activity, and compared with that in Scott syndrome. The most significant abnormality in the new patients, whose defect may differ from that in Scott syndrome, is an impairment in collagen plus thrombin-induced prothrombinase activity in the absence of added factor Va. In one of these patients this may be caused by an abnormality in platelet alpha-granule factor V distinct from that described for factor V Quebec, alpha delta-SPD, or alpha-SPD (gray platelet syndrome). Prothrombinase activity in response to all agonists was impaired in delta-SPD and was associated with an inability of these platelets to maintain elevated intracellular calcium levels. Both the rapid decline in agonist-induced [Ca2+]i levels and the impaired prothrombinase activation in delta-SPD platelets were corrected by the addition of adenosine diphosphate (ADP) after stimulation. These findings suggest that secreted ADP may play an important role in the generation of prothrombinase activity by contributing to the maintenance of a critical [Ca2+]i level necessary to maintain aminophospholipids on the outer surface of the platelet membrane, and provide evidence that dense granules may be a major source of ADP which can contribute to calcium influx in stimulated platelets. Parallel alterations, including both increases and decreases, in the [Ca2+]i and prothrombinase responses were also observed in thrombasthenia, depending on the agonist and stirring conditions. Both responses were increased in collagen-stimulated, unstirred platelets, whereas an inability to maintain increased [Ca2+]i levels, associated with decreased prothrombinase activity in all but one atypical patient, was seen in stirred collagen plus thrombin-activated platelets. Although the parallel alterations in these responses in thrombasthenia, as in SPD, further show the close association between the generation of prothrombinase activity and the maintenance of increased intracellular Ca2+ levels, the specific role that GPIIb-IIIa may play in both these events remains unresolved. Our findings of both enhancement and inhibition of these activation-related events in thrombasthenic platelets may be related to previous conflicting reports on the promotion or inhibition of fibrin formation by GPIIb-IIIa, and could be relevant to the use of specific inhibitors of GPIIb-IIIa as antithrombotic agents. In addition, the study provides further support for the concept that the development of agents that could induce a Scott syndrome defect in normal platelets may provide a new approach to antithrombotic therapy.

Adult↗
PubMed1998

Platelets stored in a new-generation container differences between plasma and platelet additive solution II.

BACKGROUND AND OBJECTIVES: In order to preserve platelet concentrates (PCs) with high yields, a new polyolefin platelet storage container (PL 2410, 1.3L, Baxter, La Châtre, France) with increased gas permeability in combination with a larger surface area has been developed. The storage capacity was studied with platelets in plasma and platelets additive solution. MATERIALS AND METHODS: Platelet concentrate pools (PCs) of different yields suspended in either plasma (PCs-PL; n = 30) or PAS II (PCs-PAS; n = 37) were prepared. For preparation of PCs with a low, intermediate and high number of platelets 3, 5 and 6 buffy coat (BCs) were pooled with different volumes of plasma and 5 and 6 BCs were pooled with different volumes of PAS II, in order to obtain PCs of equal volumes comparable with routine conditions. All PCs were stored on a flatbed shaker at 22+/-2 degrees C and evaluated on days 1, 2, 5 and 7 by determining platelet and white cell counts, pH (37 degrees C), pO2, pCO2 and swirling score. RESULTS: Platelet yields ranged from 1.5 up to 5.5 x 10(11) platelets/U. On day 7 all PCs-PL (n = 4) with platelet yields above 4.5 x 10(11)/U had a pH value below 6.8 (range 5.91-6.79). While 7 of 8 PCs-PAS units with platelet yields above 4. 0 x 10(11)/U showed a pH value below 6.8 (range 6.31-6.70). CONCLUSION: During 7 days of storage in a new 1.3-liter platelet container, the pH was maintained above 6.8 in PCs in plasma with a yield between 1.5- and 4.5 x 10(11)/U; when PAS II was used, the maximum platelet yield allowed for comparable pH maintenance was somewhat lower (4.0 x 10(11)/U).

Adult↗
PubMed2001

Use of ICHOR-platelet works to assess platelet function in patients treated with GP IIb/IIIa inhibitors.

Platelet glycoprotein GP IIb/IIIa inhibitors have been recently approved for use in treating patients with acute coronary syndromes and those undergoing PCI. The purpose of this study was to assess the feasibility of using a new device, the ICHOR platelet works, to detect platelet inhibition in patients undergoing PCI and treated with abciximab or tirofiban. The study was conducted at Baylor College of Medicine, Houston, Texas. Thirty patients undergoing PCI and treated with abciximab (n = 10) or tirofiban (n = 20) are included. Blood samples were obtained before, at 30 min, at 4 hr, and at 12 hr after starting the GP IIb/IIIa inhibitors and 2 hr after discontinuation. Baseline studies revealed > 95% platelet aggregability in all patients after exposure to ADP (20 microM). After starting tirofiban, 82%, 83%, and 82% of platelets were inhibited at 30 min, 4 hr, and 12 hr. Platelet inhibition decreased to 43% 2 hr after discontinuation of tirofiban. Similarly, ICHOR platelet works detected 91%, 92%, and 85% platelet inhibition at 30 min, 4 hr, and 12 hr after starting abciximab, respectively. Platelet inhibition decreased to 73% 2 hr after discontinuation. The ICHOR platelet works is a promising, simple, and rapid bedside method that may have clinical utility in assessing platelet inhibition in patients treated with GP IIb/IIIa inhibitors. Cathet Cardiovasc Intervent 2001;53:346-351.

Abciximab↗
PubMed2001

Testing platelet activation with a shear-dependent platelet function test versus aggregation-based tests: relevance for monitoring long-term glycoprotein IIb/IIIa inhibition.

BACKGROUND: Tests developed to monitor glycoprotein (GP) IIb/IIIa blockade do not properly reflect platelet function in vivo and need a baseline (pretreatment) value. Because GP IIb/IIIa is essential in platelet aggregation and thrombosis under shear conditions, a flow-dependent approach to monitor its inhibition can be used. METHODS AND RESULTS: We compared a test based on flow-dependent platelet deposition, the Cone and Platelet Analyzer (CPA), with in vitro platelet aggregometry and the Rapid Platelet Function Assay (RPFA) on platelet function after GP IIb/IIIa inhibition. In vitro, increasing concentrations of abciximab (0% to 100% receptor occupancy) were tested. Ex vivo, platelet function was monitored with the CPA and with aggregometry for up to 1 week after abciximab administration. The CPA was better correlated with the percentage of free GP IIb/IIIa receptors than was aggregometry or the RPFA. Only the RPFA, when expressed as a ratio over baseline (pretreatment), was comparable to the CPA. Ex vivo, the CPA, but not aggregometry, showed prolonged platelet inhibition with gradual recovery from GP IIb/IIIa receptor blockade in the first week after abciximab administration. CONCLUSIONS: Platelet function assessment by shear-induced deposition is a reliable test to monitor a wide range of GP IIb/IIIa inhibition. Its accuracy does not require a baseline reference. The effects of GP IIb/IIIa blockade on platelet function should be examined under high shear conditions.

Abciximab↗
PubMed1983

A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.

To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.

Animals↗
PubMed2006

Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents.

GPVI is a 62-kDa membrane glycoprotein expressed in noncovalent association with the Fc receptor gamma chain on human and murine platelets and serves as the major activating receptor for collagen. GPVI-specific antibodies have the capacity to specifically deplete GPVI from mouse and human platelets in vivo, rendering them unresponsive to collagen and GPVI-specific agonists. Such antibodies do not remove GPVI from noncirculating platelets in vitro, however, making it difficult to evaluate their antithrombotic potential and mechanism of action, particularly in human platelets. We devised a model system in which human platelets are introduced into the retroorbital plexus of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, allowed to circulate, and evaluated for the effects of GPVI-specific murine monoclonal antibodies (mAbs) on platelet survival and function. GPVI-specific mAbs triggered depletion of GPVI from human, but not murine, platelets. Soluble truncated human GPVI appeared concomitantly in mouse plasma. GPVI-depleted human platelets had markedly diminished responses to GPVI-specific agonists and unexpectedly exhibited somewhat depressed responses to G-protein-coupled agonists. The ability to evaluate in living mice the in vivo function and survival of circulating human platelets may prove valuable for determining mechanisms of antibody-mediated platelet passivation and aid in the development of novel anti-platelet therapeutics.

Animals↗