EDTA-induced changes in platelet structure and function: clot retraction

Ethylenediamine tetracetic acid (EDTA) is known to cause structural, biochemical and functional injury to blood platelets, including irreversible dissociation of the fibrinogen receptor, glycoprotein alpha IIb beta 3 (GPIIb/IIIa). Despite inability to adhere to glass, spread, and to aggregate in response to adenosine diphosphate (ADP) and other agonists, EDTA-treated platelets support clot retraction as well as untreated cells. The present study has used clot retraction under isometric tension and electron microscopy to determine if there are any differences in platelet-fibrin interactions of clots formed from blood collected in EDTA or platelets from blood drawn into citrate (CCD) anticoagulants. No physical differences could be identified. Polymerizing fibrin bound intimately to aggregates developing from EDTA platelets undergoing shape change, internal transformation, adhesion and spreading on fibrin strands oriented in the long axis of contraction. The results suggest that reassociation of irreversibly dissociated GPIIb/IIIa takes place immediately after initiation of clot retraction, or that a significant proportion of GPIIb/IIIa receptors on resting platelets are inaccessible to EDTA and become available after activation by thrombin.     

Comparative in vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet-mediated clot strength induced by tissue factor with use of thromboelastography: differentiation among glycoprotein IIb/IIIa antagonists

In the present study, the in vitro efficacy of different platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists on platelet-fibrin-mediated clot strength under shear was compared with the antiaggregatory efficacy by using tissue factor (TF) thromboelastography (TEG). The ability of platelets to augment the elastic properties of blood clots under shear conditions was measured by computerized TEG under conditions of maximal platelet activation accelerated by recombinant TF. Under these conditions, platelets significantly enhance clot strength 8-fold (relative to platelet-free fibrin clots). This effect was inhibited to a different extent by various platelet GPIIb/IIIa receptor antagonists; this inhibition appears to be dependent on the transmission of platelet contractile force to fibrin via the GPIIb/IIIa receptors. The GPIIb/IIIa antagonists with high binding affinity for resting and activated platelets and slow platelet dissociation rates (class I) but not those with fast platelet dissociation rates (class II) demonstrated potent and comparable inhibition of platelet aggregation and TF-TEG clot strength. Platelet GPIIb/IIIa antagonists of class I, such as XV459 (free-acid form of roxifiban), DMP802, XV454, and c7E3, demonstrated comparable inhibitory dose responses of TF-TEG clot strength and platelet aggregation, with an IC(50) of 50 to 70 nmol/L. In contrast, platelet GPIIb/IIIa antagonists from class II, with comparable antiaggregatory efficacy, such as DMP728, YZ202 (free-acid form of orbofiban), YZ211 (free-acid form of sibrafiban), YZ751, and other antagonists, have a much lower efficacy in altering the strength of TF-mediated clot formation (IC(50) >1.0 micromol/L). These data suggest differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-fibrin clot retraction despite of equivalent antiaggregatory potency.     

Differential efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet/fibrin-mediated clot dynamics under different conditions using thrombelastography: the critical need for anticoagulant

BACKGROUND: Intravenous GpIIb/IIIa antagonists demonstrate various significant clinical benefits depending on the agent used. In contrast, oral delivery of GpIIb/IIIa antagonists failed in achieving clinical benefits. This raises the question about the differences among different GpII/IIIa antagonists. METHODS: The effect of various platelet glycoprotein (GP) IIb/IIIa antagonists on the dynamics of platelet/fibrin clot formation and strength was determined using thrombelastography under different conditions. RESULTS: GPIIb/IIIa antagonists with high affinity for resting and activated platelets and with slow rates of dissociation from GPIIb/IIIa (Class I antagonists) demonstrated potent and comparable inhibition of platelet aggregation and platelet-mediated clot strength under different conditions. In contrast to antagonists that dissociate rapidly from GPIIb/IIIa (class II antagonists). Class I antagonists such as the free acid form of roxifiban inhibited platelet-mediated clot strength, with the inhibiting concentration required for 50% effect (IC50) = 70 n mol/l, whereas the IC50 of the class II antagonists such as the free acid forms of orbofiban, sibrafiban, lotrafiban, integrilin or aggrastat ranged from 1 to 15 micromol/l. The IC50s for class II antagonists in inhibiting platelet/fibrin clot formation and strength were substantially greater (10-15 fold) than their clinically achievable concentrations. The limited efficacy for class II antagonists in inhibiting platelet-mediated clot dynamics was enhanced by the combination with heparin. CONCLUSIONS: Thus, these data indicated that there are differences in the efficacy of various GPIIb/IIIa antagonists in inhibiting platelet/fibrin clot formation and strength, which might be corrected by heparin. Data also suggest that inhibition of platelet aggregation may not be the sole determinant for the in-vivo efficacy of various GPIIb/IIIa antagonists.     

EDTA-induced changes in platelet structure and function: adhesion and spreading

Ethylenediamine tetraacetic acid (EDTA) is an effective anticoagulant, but unfortunately causes structural, biochemical and functional damage to human platelets. Some of the functional injuries, such as adhesion to and spreading on surfaces, are considered irreversible. The present investigation has evaluated that hypothesis. Our findings indicate that platelets from EDTA platelet-rich plasma (PRP) or CCD PRP to which EDTA has been added do not adhere to glass or plastic surfaces. However, when platelets from EDTA PRP or CCD PRP containing added EDTA are washed and resuspended under conditions reported to cause irreversible dissociation ofthe fibrinogen receptor, GPIIb/IIIa, then washed and resuspended in buffer containing Ca 2+ and Mg 2+ ions will adhere and spread in the same manner as platelets not exposed to EDTA. The ability to recover adhesive function may explain why EDTA platelets are able to sustain clot retraction as well as CCD platelets.     

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann''s thrombasthenia

A patient is described with a disturbance of platelet function comparable to that in Glanzmann's thrombasthenia. Platelet aggregation and binding of fibrinogen to the patient's platelets were defective and thrombin-induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex-dependent anti-GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22 degrees C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two-dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.     

EDTA-induced changes in platelet structure and function: adhesion and spreading

Ethylenediamine tetraacetic acid (EDTA) is an effective anticoagulant, but unfortunately causes structural, biochemical and functional damage to human platelets. Some of the functional injuries, such as adhesion to and spreading on surfaces, are considered irreversible. The present investigation has evaluated that hypothesis. Our findings indicate that platelets from EDTA platelet-rich plasma (PRP) or CCD PRP to which EDTA has been added do not adhere to glass or plastic surfaces. However, when platelets from EDTA PRP or CCD PRP containing added EDTA are washed and resuspended under conditions reported to cause irreversible dissociation of the fibrinogen receptor, GPIIb/IIIa, then washed and resuspended in buffer containing Ca2+ and Mg2+ ions will adhere and spread in the same manner as platelets not exposed to EDTA. The ability to recover adhesive function may explain why EDTA platelets are able to sustain clot retraction as well as CCD platelets.     

Comparative efficacy of different low-molecular-weight heparins (LMWHs) and drug interactions with LMWH: implications for management of vascular disorders

The low-molecular-weight heparins (LMWHs) are more efficacious and safer than unfractionated heparin (UFH) in the prevention and treatment of venous thrombosis and to a certain extent in the treatment of acute ischemic syndromes. Because of their predictable pharmacokinetics and bioavailability after subcutaneous administration, LMWHs can be more convenient for outpatient use than UFH. Differences in the manufacturing process of LMWHs result in significant structural and molecular weight differences; thus, LMWHs have individual biochemical and pharmacological profiles and are not interchangeable on the basis of either mass or anti-Xa activity. Using thromboelastograph (TEG) and platelet aggregometry, this investigation compared the in vitro efficacy among various LMWHs and examined the interactions between LMWHs and platelet glycoprotein (GP) IIb/IIIa antagonists. TEG was used to determine the ability of platelet and fibrin interactions to augment blood clots, an effect measured under conditions of maximal platelet activation during clot formation accelerated by recombinant human tissue factor (TF). The comparative efficacy of LMWHs on different mediator-induced clot retraction in human blood was assessed by TEG, which demonstrated the potency of different LMWHs to inhibit various mediator-induced clot formations under shear. Tinzaparin was relatively more effective in inhibiting TF-, lipopolysaccharide-, factor (f) Xa-, and thrombin-induced clot formation under shear. Under these conditions, platelets significantly enhance clot strength (eightfold vs. platelet-free fibrin clots). LMWHs appear to have broader efficacy than other anticoagulants. Abciximab and roxifiban further inhibited clot strength by affecting the transmission of platelet contractile force to fibrin by platelet GPIIb/IIIa receptors. Subtherapeutic doses of tinzaparin combined with abciximab or roxifiban resulted in a distinct synergy that improved anticoagulant and antiplatelet efficacy mediated by TF, fXa, or thrombin. As these data suggest, the combination of low-dose tinzaparin with low-dose GPIIb/IIIa antagonists (abciximab, roxifiban) may be efficacious in the prevention and treatment of various thromboembolic disorders.     

In-vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists and thrombolytics on platelet/fibrin-mediated clot dynamics in human whole blood using thrombelastography.

Suppressing platelet activation improves efficacy of thrombolytic therapy for stroke and acute myocardial infarction. Combination treatment with recombinant tissue plasminogen activator (r-tPA) and glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitor that binds with high affinity to platelets may therefore improve the efficacy of thrombolytic therapy. The effect of platelet GPIIb/IIIa antagonists and/or r-tPA on the dynamics of platelet/fibrin clot formation, strength, and lysis was determined using thrombelastography in human blood under thrombin or tissue factor stimulation. The study utilized platelet GPIIb/IIIa antagonists with high affinity and slow off-rate (Class I) from resting and activated platelets in comparison with Class II antagonists (lower affinity and fast off-rate from platelet GPIIb/IIIa receptors). The combination of the active form of roxifiban (XV459; Class I) or the active form of orbofiban (Class II) with a subeffective concentration of r-tPA resulted in a synergistic effect in clot lysis with roxifiban active form XV459 but not with that of orbofiban at therapeutically achievable concentrations that inhibit human platelet aggregation. These data indicate differential enhanced thrombolysis of low levels of r-tPA with high-affinity Class I but not with low-affinity Class II GPIIb/IIIa antagonists in the absence of anticoagulants.     

von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets

We have investigated two major questions related to the molecular basis of interactions between the three-dimensional fibrin network and thrombin-stimulated human platelets. First, what are the roles played by glycoproteins (GP) Ib and IIb:IIIa in linking the fibrin clot tightly to the platelet surface? Second, does von Willebrand factor (vWF) modulate the extent of platelet-fibrin interactions? Quantitative fluorescence microscopy (microfluorimetry) has been used to determine the quantity of fluorescein-labeled fibrin bound to the surface of thrombin-stimulated, gel-filtered platelets (the supernatants of which contained small quantities of vWF) in the presence/absence of receptor-specific and vWF-specific monoclonal antibodies (MoAbs), as well as exogenous vWF. A MoAb specific for the GPIIb:IIIa complex exhibited a concentration-dependent inhibition of fibrin binding, whereas a MoAb specific for GPIb was ineffective in this regard. Similarly, a MoAb that recognizes the N-terminal region of vWF involved in GPIb binding did not influence fibrin binding. In contrast, a MoAb that binds to a C-terminal region of vWF involved in GPIIb:IIIa recognition caused a specific, concentration-dependent increase in the quantity of platelet-bound fibrin. We also found that exogenous vWF caused a concentration-dependent decrease in fibrin binding. These results support the hypothesis that vWF and fibrin, both of which are multimeric adhesive ligands, compete for occupancy of the GPIIb:IIIa complex on thrombin-stimulated platelets.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

P-selectin, tissue factor and CD40 ligand expression on platelet-leucocyte conjugates in the presence of a GPIIb/IIIa antagonist

This study was to investigate the appearance of P-selectin, tissue factor (TF) and CD40 ligand (CD40L) on platelet-leucocyte conjugates in the absence and presence of a GPIIb/IIIa antagonist, MK-852, and the effect of adding EDTA to pre-formed conjugates. The purpose was to find out whether these antigens are displaced from the conjugates along with the platelets, thus providing information on their location. Hirudinized blood was stirred with collagen ((2 microg/mL) in the absence and presence of MK-852 (10 micromol/mL)). P-selectin, TF and CD40L were measured on platelet-leucocyte conjugates (CD42a positive monocytes and neutrophils) and on single platelets by flow cytometry. Measurements were also made after subsequent addition of EDTA (4 mmol/L). Platelet-leucocyte conjugate formation was markedly enhanced in the presence of MK-852. P-selectin, TF and CD40L expression on the conjugates was also enhanced. Monocytes bound more platelets and expressed more P-selectin, TF and CD40L than neutrophils. EDTA displaced the majority of platelets from the conjugates and also the P-selectin, TF and CD40L, whereas it did not displaced P-selectin or CD40 ligand from the platelets themselves. It is concluded that a GPIIb/IIIa antagonist promotes formation of platelet-leucocyte conjugates, which display P-selectin, TF and CD40L that appears to be associated with the adherent platelets. Platelet-monocyte conjugates are prime candidates for arterial inflammation and thrombosis. Pro-inflammatory and pro-thrombotic effects of CD40L and tissue factor may be an explanation of the negative clinical effects using GPIIb/IIIa antagonists.     

P-selectin,tissue factor and CD40 ligand expression on platelet-leucocyte conjugates in the presence of a GPIIb/IIIa antagonist

This study was to investigate the appearance of P-selectin, tissue factor (TF) and CD40 ligand (CD40L) on platelet-leucocyte conjugates in the absence and presence of a GPIIb/IIIa antagonist, MK-852, and the effect of adding EDTA to pre-formed conjugates. The purpose was to find out whether these antigens are displaced from the conjugates along with the platelets, thus providing information on their location. Hirudinized blood was stirred with collagen ((2?μg/mL) in the absence and presence of MK-852 (10?μmol/mL). P-selectin, TF and CD40L were measured on platelet-leucocyte conjugates (CD42a positive monocytes and neutrophils) and on single platelets by flow cytometry. Measurements were also made after subsequent addition of EDTA (4?mmol/L). Platelet-leucocyte conjugate formation was markedly enhanced in the presence of MK-852. P-selectin, TF and CD40L expression on the conjugates was also enhanced. Monocytes bound more platelets and expressed more P-selectin, TF and CD40L than neutrophils. EDTA displaced the majority of platelets from the conjugates and also the P-selectin, TF and CD40L, whereas it did not displaced P-selectin or CD40 ligand from platelets themselves. It is concluded that a GPIIb/IIIa antagonist promotes formation of platelet-leucocyte conjugates, which display P-selectin, TF and CD40L that appears to be associated with the adherent platelets. Platelet-monocyte conjugates are prime candidates for arterial inflammation and thrombosis. Pro-inflammatory and pro-thrombotic effects of CD40L and tissue factor may be an explanation of the negative clinical effects using GPIIb/IIIa antagonists.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

G?r?g Thrombosis Test: a global in-vitro test of platelet function and thrombolysis.

This is the first laboratory evaluation of a new instrument, designed to test both platelet function and thrombolytic activity from a native blood sample, in vitro. The inventor assumed that the reduction and arrest of blood flow was due to activation, aggregation and stabilized thrombus formation by shear-activated platelets, and that re-establishment of flow was due to thrombolysis. Morphologic and functional studies presented here confirm these mechanisms. In vitro tests provided incontestable evidence for the principal role of platelets in the obstruction of flow (occlusion time) and for thrombolysis as the principal mechanism underlying the restoration of blood flow (lysis time). In addition to aggregation, it is the explosive generation of thrombin by shear-activated platelets that results in the formation of an occlusive haemostatic thrombus. Anticoagulation of blood completely prevented occlusion. Platelet-rich thrombus formation (occlusion time) was dose-dependently inhibited by monoclonal antibody against platelet glycoprotein (GP) Ib (6B4 and 12E4), aurin tricarboxylic acid, monoclonal antibody against platelet GPIIb/IIIa (MA-16N7C2 and abciximab), a synthetic GPIIb/IIIa antagonist (TAK-029), thrombin inhibitor (argatroban), and anti-von Willebrand factor, but not by anti-fibrinogen. Plasminogen activator streptokinase (Varidase) and tissue-type plasminogen activator (Monteplase) dose-dependently enhanced thrombolysis (lysis time) without affecting platelet function (occlusion time). The test is specific for thrombolysis. The plasmin inhibitor tranexamic acid prevented plasminogen activator-induced thrombolysis, while inhibition of clot retraction by cytochalasin B did not affect the lysis time. This rapid and sensitive global test of platelet function and thrombolytic activity could be of great value both in research and in clinical practice.     

Type I Glanzmann thrombasthenia: most common subtypes in North Indians

The expression of GPIIb/IIIa on the platelet surface was assessed in 10 patients with Glanzmann thrombasthenia and their families by flow cytometry to determine the common subtype in North Indians. Glanzmann thrombasthenia was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to ADP, ADR, arachidonic acid, and collagen. Flow cytometry revealed variable GPIIb/IIIa expression by CD61 and CD41 in patients with Glanzmann thrombasthenia on the basis of CD61 levels, six patients were subtyped as type I because they had absent GPIIb/IIIa, three patients were subtyped as type II because their GPIIb/IIIa levels varied from 7.72% to 20.40%, and one patient was diagnosed as type III, because his clot retraction was 60% and GPIIb/IIIa was 46.0% of normal. Four fathers, three mothers, and five siblings were found to have GPIIb/IIIa levels less than 35% of normal. It is possible that low GPIIb/IIIa levels in family members may reflect their carrier status. It is postulated that flow cytometric estimation of GPIIb/IIIa in parents/siblings may detect carrier status in Glanzmann thrombasthenia.     

Differential antiplatelet efficacy for various GPIIb/IIIa antagonists: role of plasma calcium levels

OBJECTIVES: The present study was undertaken to determine the effects of free ionized calcium influenced by either the anticoagulant used (citrate vs. heparin) or directly varying the calcium levels after treatment of blood with citrate on the antiplatelet efficacy of two classes of GPIIb/IIIa antagonists. METHODS: The platelet effects of changes in plasma [Ca(++)] with the different GPIIb/IIIa antagonists were determined using light transmittance aggregometry, direct binding kinetics, and (125)I-fibrinogen binding to activated human platelets. RESULTS: A significantly higher IC50s was shown with heparin (free ionized calcium=1.1 mM) as compared to that with citrate (free ionized calcium=0.12 mM) with class II GPIIb/IIIa antagonists (P<0.01) such as Orbofiban, and Integrilin. In contrast, class I GPIIb/IIIa antagonists such as Roxifiban and Abciximab showed no significant changes in their IC50s in either citrate or heparin. Similar data were shown with other non-calcium chelating anticoagulant such as PPACK as compared to that with heparin. Additionally, similar data were shown with regard to the [Ca(++)] sensitivity for GPIIb/IIIa antagonists from Class II but not Class I in the changes in IC50 values required for the inhibition of (125)I-fibrinogen binding to activated human gel filtered platelets. Additionally, examples from Class I GPIIb/IIIa antagonists such as (3)H-active form of Roxifiban showed no significant changes in its platelet binding affinity in response to change in [Ca(++)]. In contrast, GPIIb/IIIa antagonists from class II such as (3)H-active form of Orbofiban demonstrated significant changes (P<0.01) in its platelet binding kinetics and antiplatelet efficacy in response to changes in Ca(++) concentrations. CONCLUSIONS: These data suggest the impact of the method of blood collection or changes in plasma calcium levels on the antiplatelet efficacy for class II but not class I GPIIb/IIIa antagonists depending on their platelet binding kinetics.     

The Membrane Glycoprotein IIb/IIIa Complex Mediates Deposition of Thrombin-stimulated Blood Platelets on Polystyrene Plastic Under Static Conditions

A major challenge in the use of artificial materials for implant devices, artificial organs, and extra-corporeal circulation systems, is the adhesion of platelets and the subsequent formation of platelet aggregates on the non-biological surface. The mechanism of platelet attachment to artificial surfaces is not completely understood. Using an enzyme immunoassay, we examined platelet deposition to the polystyrene plastic of microtiter plate wells under static conditions. Following thrombin stimulation, platelets adhered to the wells. This adhesion process was suppressible by the use of different substances known to interfere with the function of the platelet surface glycoprotein IIb/IIIa complex (GPIIb/IIIa). The substances we used were ethylenediaminetetraacetic acid (EDTA), tetrapeptide RGDS (Arg-Gly-Asp-Ser), and a monoclonal antibody directed against the IIIa moiety of the GPIIb/IIIa complex. Our results indicate that the GPIIb/IIIa complex is the platelet receptor which mediates platelet adhesion to polystyrene plastic under such static conditions. The GPIIb/IIIa complex should consequently be regarded as a multifunctional platelet regulator which, depending on the circumstances, may support platelet adhesion as well as platelet aggregation. By contrast, a monoclonal antibody directed against the platelet surface glycoprotein complex Ib/IX (GPIb/IX) did not under the same static conditions inhibit platelet deposition to the polystyrene plastic. In the microtiter wells, platelet alpha-granular proteins were detected either on the surface of adherent platelets or, when platelet deposition was inhibited by EDTA directly on the polystyrene plastic. In the latter case, fibrinogen and thrombospondin were definitely the dominating proteins. The presence of platelet-derived proteins in the microtiter wells significantly enhanced the adhesion of thrombin-stimulated platelets but not of non-stimulated platelets.     

Effects of different blood components on clot retraction analysed by measuring elasticity with a free oscillating rheometer

Free oscillation rheometry (FOR) using the ReoRox 4 instrument makes it possible, at bedside, to study the coagulation process in blood over time and gives information on clotting time and coagulum elastic properties. In order to find out how various factors influence the FOR analysis we studied the coagulation process and change of elasticity over time in non-anticoagulated and citrated blood samples, plasma samples with various platelet concentrations (0-200 x 109/l) and blood samples with various haematocrit (0-40%). Blood samples supplemented with fibrinogen were analysed to elucidate the importance of fibrinogen on elasticity. The importance of the GPIIb/IIIa receptor on platelets was investigated by comparing the elasticity development in blood samples in presence and absence of a GPIIb/IIIa receptor inhibitor, abciximab. Anticoagulation with citrate did not have major influence on the viscoelastic properties of the coagulum. Increasing number of platelets and increasing fibrinogen concentration resulted in higher elasticity while increasing haematocrit gave lower elasticity. Blood samples with GPIIb/IIIa receptor inhibitor had very low elasticity indicating the importance of functional GPIIb/IIIa receptors. In conclusion we consider FOR to be a useful method to study the elastic properties of the coagulum. Various factors such as the number of red blood cells and platelets as well as the fibrinogen concentration should be taken into consideration when evaluating the results. The ReoRox 4 instrument had excellent measuring range and unusually small artefactual effects on clot elasticity induced by the instrument in comparison with published results on other instruments.     

Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation

Tissue plasminogen activator (TPA) converts plasminogen to plasmin within the fibrin clot, thus localizing activation of fibrinolysis. To determine the extent to which platelets promote activation of plasminogen by TPA, we studied the interaction of TPA and plasminogen with unstimulated platelets. Normal washed platelets incubated in the presence of physiologic concentrations of plasminogen (180 micrograms/mL) and TPA (20 ng/mL) failed to generate plasmin activity. In contrast, incubation of platelets with TPA concentrations achieved during thrombolytic therapy (40 to 800 ng/mL) produced a tenfold to 50- fold increase in plasmin activity. After exposure to plasminogen and 200 ng/mL of TPA for one hour, platelets failed to agglutinate in the presence of ristocetin. Incubation of platelets suspended in autologous plasma with 400 ng/mL of TPA for one hour also inhibited ristocetin- induced agglutination. Exposure of platelets to plasminogen and increasing concentrations of TPA correlated with a decrease in glycoprotein Ib (GPIb) and an increase in glycocalicin, as shown by immunoblotting. The glycoprotein IIb/IIIa (GPIIb/IIIa) complex and a 250,000-dalton protein also disappeared from washed platelets after incubation with plasminogen and 200 ng/mL of TPA for one hour. These platelets failed to aggregate in the presence of adenosine diphosphate (ADP) or gamma thrombin, although aggregation in response to calcium ionophore A23187 and arachidonic acid remained intact. However, aggregation in response to all four agonists was normal when platelets were incubated with TPA in the presence of autologous plasma. Platelets from a patient with Glanzmann's thrombasthenia also generated plasmin in the presence of TPA. Hydrolysis of GPIb and inhibition of ristocetin- induced agglutination occurred to a lesser extent with these platelets than with control platelets. We conclude that platelets provide a surface for activation of plasminogen by pharmacologic amounts of TPA. Plasmin generation leads to degradation of GPIb and decreased ristocetin-induced agglutination in normal and thrombasthenic platelets, as well as degradation of GPIIb/IIIa in normal washed platelets and inhibition of ADP and gamma thrombin-induced aggregation. These findings suggest that pharmacologic concentrations of TPA may cause platelet dysfunction due to plasmin generation on the platelet surface.