Reassociation and translocation of glycoprotein IIB-IIIA in EDTA-treated human platelets

Platelet membrane glycoproteins IIb and IIIa form a calcium-dependent heterodimer that plays a key role in platelet adhesion and aggregation. The present objective was to measure the dissociation and reassociation of GPIIb-IIIa by flow cytometric analysis of platelets labelled with mAbs specific for the glycoprotein complex or each monomer. In agreement with previous studies, EDTA chelation of extracellular calcium, [Ca2+]o, dissociated the heterodimer in a time and temperature dependent manner. Agonist stimulation of EDTA-treated platelets induced subunits to reassociate with the following order of potency: thrombin > collagen > ADP. Two-fold increases in GPIIb-IIIa and GPIIb indicate that thrombin caused reassociation of surface subunits and concurrent translocation of complexes from intracellular pools. The latter was partially inhibited by cytochalasin B thus indicating that a subpopulation of GPIIb-IIIa required cytoskeletal remodelling for translocation. Surface GPIIIa as reported by anti-CD61 declined more and upregulated less compared with GPIIb-IIIa or GPIIb. Results suggest that EDTA incubation might have altered the conformation of this epitope and decreased mAb binding. Collagen induced GPIIb-IIIa reassociation but not translocation of cryptic complexes. BAPTA suppression of rises in cytosolic calcium concentration or low [Ca2+]o inhibited GPIIb-IIIa reassociation, thus indicating that this reaction was driven by signal transduction. Thrombin and collagen induced a comparable level of aggregation of EDTA-treated platelets despite a 3-fold difference in cell surface GPIIb-IIIa. It is concluded that the effects of EDTA on GPIIb-IIIa dissociation and loss of adhesive functions are largely but not completely reversible.     

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.     

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.     

Glycoprotein IIb and IIIa retention on fibrinogen-coated surfaces after lysis of adherent platelets

The platelet-membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex is essential for platelet aggregation and is involved in the attachment of platelets to thrombogenic surfaces. This study shows the retention of GPIIb and GPIIIa on immobilized fibrinogen after Triton X-100 (Sigma Chemical Co, St Louis, MO) lysis of adherent platelets. Glycoproteins were detected using subunit specific monoclonal antibodies in a modified enzyme-linked immunosorbent assay procedure. GPIIb-IIIa retention was judged to be specific relative to GPIb recovery, and was modulated by platelet activation. Platelet exposure to adenosine diphosphate or thrombin, but not A23187 or chymotrypsin, markedly enhanced GPIIb and GPIIIa recovery relative to that observed with unstimulated platelets, or prostaglandin E1-treated platelets. Moreover, lysis of adherent platelets in the presence of 10 mmol/L EDTA, under conditions promoting GPIIb-IIIa complex dissociation (pH 8.1, 60 minutes, 37 degrees C), had no effect on GPIIb or GPIIIa subunit recovery. Platelet activation with Zn+2 also enhanced GPIIb and GPIIIa recovery on fibrinogen-coated surfaces over that observed with unstimulated platelets, but GPIIb and IIIa retention was EDTA sensitive. This correlated with the EDTA-reversible nature of Zn+2- activated platelet adhesion to fibrinogen-coated surfaces. The data (1) show that platelet adhesion to fibrinogen is accompanied by the induction of high-affinity interactions between GPIIb-IIIa and immobilized fibrinogen that are EDTA-resistant and enhanced by platelet activation with some but not all agonists, and (2) implicate these interactions in stabilizing platelet contacts with fibrinogen-coated surfaces.     

Structural biology of glycoprotein IIb-IIIa

Glycoprotein IIb-IIIa (GPIIb-IIIa), a calcium-dependent heterodimer whose expression is restricted to platelets and megakaryocytes, contains a binding site for protein ligands such as fibrinogen and von Willebrand factor (vWf) whose exposure by platelet stimulation is a prerequisite for platelet aggregation. GPIIb-IIIa heterodimers are assembled from nascent GPIIb and GPIIIa subunits in the calcium-rich environment of the endoplasmic reticulum, and correctly folded heterodimers are transported from the endoplasmic reticulum through the Golgi apparatus to the cell surface. Agonist stimulation of platelets produces a conformational change in GPIIb-IIIa that exposes its ligand binding site, a process termed "insideout" signaling. This signaling process, by interacting with the cytoplasmic extensions of GPIIb and GPIIIa, converts the heterodimer from an inactive to an activated state capable of binding soluble ligands.     

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A(2) analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.     

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.     

Detection of an epitope specific for the dissociated form of glycoprotein IIIa of platelet membrane glycoprotein IIb-IIIa complex and its expression on the surface of adherent platelets

Summary Glycoproteins (GPs) IIb and IIIa form a Ca²⁺-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb). CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate. (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1–100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca²⁺ and Mg²⁺, 2.34 ± 0.28 ± 10³ molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 ± 0.28 ± 10³ molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However. binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using ⁵¹Cr-labelled platelets, binding of ^l25I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 ± 0.48 ± 10³ per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa: (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.     

An echistatin-like Arg-Gly-Asp (RGD)-containing sequence in the heavy chain CDR3 of a murine monoclonal antibody that inhibits human platelet glycoprotein IIb/IIIa function

We describe the production and biochemical characterization of the first GPIIb/IIIa-inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immunoaffinity-purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA-16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet-activating factor, whereas ristocetin-induced aggregation was unaffected. MA-16N7C2 (IgG2a) bound ˜ 4 times faster to activated than to resting platelets, with a K_dcalc of 6.6 nm and of 17.5 nm , respectively. Equilibrium binding studies to non-activated platelets showed a K_d of 18.2 n_m with 41 x 10³ binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca²⁺ -dependent complex. MA-16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a K_i of 8.5 nm and 13.2 nm , respectively. Sequence analysis revealed a RGD-containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain. That this RGD-containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.     

Changes in Free Calcium Concentrations in Platelets of SHRSP and WKY: Its Relationship to ATP Releasing Potencies and Platelet Aggregation Activities Upon Stimulation of Several Reagents

To clarify the relationships between free calcium levels, ATP release and aggregation potencies of SHRSP platelets, we examined the platelets from 9-month-old SHRSP and WKY using fura-2AM and luciferine-luciferase. In the absence of extracellular Ca²⁺, each reagent elevated the free calcium level to the same extent in the samples of both SHRSP and WKY. With regard to ATP release, thrombin and collagen less potentiated the platelet action in SHRSP than WKY, and ATP release was not affected by extracellular Ca²⁺. Collagen and ADP induced aggregations showed lower activities in SHRSP than WKY. TPA caused higher Ca²⁺ influx and aggregation activity in SHRSP than WKY in the presence of extracellular Ca²⁺. These results indicate that Ca²⁺ release must be followed by ATP release, and ATP release may be less potentiated, while thrombin and TPA induced aggregation is likely to be stimulated in SHRSP platelets, because protein kinase C activity in SHRSP platelets appears to be high.     

Aggregation of Human Platelets Stimulates Calcium Ion Movement and Release Reaction

We studied the effect of inhibition of platelet aggregation (obtained by omitting sample stirring and by the addition of ASA and GRGDS peptide) on ATP secretion and Ca²⁺ movements. When collagen was the agonist, platelet aggregation stimulated release reaction and Ca²⁺ movements in both the presence and absence of extracellular Ca²⁺. When platelets were stimulated by thrombin, ATP release and Ca²⁺ movements were largely independent of aggregation. Our data suggest that platelet aggregation stimulates Ca²⁺ movement, and that this phenomenon of feedback amplification of platelet activation plays an important role in platelet function when collagen is the agonist, while it has little or no role when thrombin is the stimulus.     

Cilostazol inhibits platelet-leukocyte interaction by suppression of platelet activation

The influence of three anti-platelet drugs, cilostazol, aspirin, and tirofiban, was investigated on platelet-leukocyte interaction by flow cytometry. When platelets and leukocytes were pre-incubated with anti-platelet drugs and stimulated by thrombin or collagen, cilostazol was found to inhibit platelet adhesion to monocytes and polymorphonuclear cells (PMNs). Similar effects were observed with anti-CD62P antibody, while aspirin and tirofiban did not appear to interfere with interaction between platelets and leukocytes. In the platelets pre-incubated with anti-platelet drugs, cilostazol significantly reduced CD62P expression and GPIIb/IIIa activation on platelet surface stimulated by thrombin or collagen. Aspirin inhibited CD62P expression and GPIIb/IIIa activation induced by collagen, but not thrombin. Tirofiban significantly blocked GPIIb/IIIa activation induced with both, and weakly inhibited CD62P expression induced by collagen. When added after stimulation of platelets, cilostazol again significantly inhibited CD62P expression and GPIIb/IIIa activation, although to a lesser extent than in the pre-incubation study. Aspirin hardly inhibited CD62P expression or GPIIb/IIIa activation, while tirofiban strongly blocked GPIIb/IIIa activation induced by thrombin or collagen, but had little effects on CD62P expression. In conclusion, our results suggest that cilostazol inhibits platelet-leukocyte interaction by reducing CD62P expression on the platelet surface.     

Inhibitory effect by new monocyclic 4-alkyliden-beta-lactam compounds on human platelet activation

In the present study some new beta-lactam compounds were screened for their ability to inhibit human platelet activation. In particular four compounds differing in the group on the nitrogen atom of the azetidinone ring were investigated. A beta-lactam having an ethyl 2-carboxyethanoate N-bound group was demonstrated to inhibit, in the micromolar range, both the Ca²⁺ release from endoplasmic reticulum, induced either by thrombin or by the ATPase inhibitor thapsigargin, and the Ca²⁺ entry in platelets driven by emptying the endoplasmic reticulum. The compound also inhibited the platelet aggregation induced by a variety of physiological agonists including ADP, collagen, thrombin and thrombin mimetic peptide TRAP. The beta-lactam reduced the phosphorylation of pleckstrin (apparent MW 47?kDa), elicited by thrombin but not by the protein kinase C activator phorbol ester. Accordingly it did not significantly affect the aggregation evoked by phorbol ester or Ca²⁺ ionophore. It was concluded that the beta-lactam likely exerts its anti-platelet-activating action by hampering the agonist induced cellular Ca²⁺ movements. The beta-lactam concentration, which significantly inhibited platelet activation, only negligibly affected the cellular viability. Even if it is still premature to draw definitive conclusions, the present results suggest that this new compound might constitute a tool of potential clinical interest and the starting-point for the synthesis of new more beneficial anti-thrombotic compounds.     

Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation

The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel-surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha-granules and microtubules, and a prominent, worm-like, fibrinogen-filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.     

Evaluation of the platelet storage pool deficiency in the feline counterpart of the chediak-higashi syndrome

Cats with the Chediak-Higashi (CH) syndrome have abnormal hemostasis with prolonged bleeding times and normal coagulation times. Platelet aggregation induced by serotonin, ADP, and collagen was impaired. Platelets from normal and CH cats were incubated with ¹⁴C-adenine and then gel-filtered. Gel-filtered platelets (GFP) from CH cats contained 63% of the ATP, 38% of the ADP, 100% of the Ca²⁺, and 75% of the Mg²⁵ of normal platelets. Serotonin could not be detected in CH platelets. Acid hydrolase and total platelet protein of CH platelets was similar to normal platelets. Gel-filtered platelets were treated with thrombin to induce maximal secretion. Secretion of ATP, Ca²⁺, and Mg²⁺ was 1.9%, 12.4%, and 16% respectively of normal platelets. ADP secretion by CH platelets was not detectable. The ATP/ADP ratio in the ¹⁴C-labeled metabolic pool of normal platelets was similar to that of total measured nucleotide pool of CH platelets. These findings suggest that in feline CH platelets, as in platelets from CH mink and cattle, there is storage pool deficiency that is virtually complete, and the virtual absence of ADP and 5HT may in part account for the abnormal hemostasis. Aggregation of platelets from CH cats was impaired, but these platelets did aggregate to arachidonate, serotonin-induced biphasic aggregation, and the aggregation response to ADP and collagen varied according to the amount of serotonin-induced TxB₂ formed. These findings support a major role for arachidonate in platelet activation.     

Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium- dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro- GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.     

Abstracts of papers presented at the Japanese–European Kobe Symposium on Thrombosis, 6–7 November 2000, Kobe Gakuin University,Kobe, Japan

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca²⁺. We have studied the involvement of exogenous Ca²⁺ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca²⁺ levels ([Ca²⁺]_i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca²⁺ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca²⁺, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca²⁺]_i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca²⁺. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca²⁺, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca²⁺ depends on a functional cytoskeleton.     

Fluorescent Indicators Give Biased Estimates of Intracellular Free Calcium Change in Aggregating Platelets: Implication for Studies with Human von Willebrand Factor

ABSTRACT: The ratiometric fluorescent indicators Fura-2 and Indo-1 are considered optimal probes for monitoring intracellular free calcium concentration ([Ca²⁺]_i). Unique problems arise, however, in studying [Ca²⁺]_ichanges induced in platelets by von Willebrand factor (vWF). Binding of native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual [Ca²⁺]_iincrease. The present investigation examined the reliability of platelet [Ca²⁺]_imeasurements in these circumstances. Ristocetin-mediated binding of vWF to human platelets promoted a slow rise in Fura-2 fluorescence ratio. Fura-2 extrusion contributed substantially to this rise, unless blocked by probenecid. Despite this precaution, the platelets were invariably contaminated slightly with extracellular indicator. As aggregation progressively reduced the number of platelets in the spectrofluorometer beam, through settling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence. This extraneous signal accounted completely for the fluorescence ratio increase, and apparent [Ca²⁺]_irise, in response to native multimeric vWF. The same problem arose with Indo-1, whereas the single wavelength indicator Fluo-3 showed the opposite pattern of apparent [Ca²⁺]_ichanges. Thus, none of these indicators provides reliable data on [Ca²⁺]_isignals in aggregating platelets. Use of a dimeric form of vWF eliminated the problem of platelet aggregates settling out of suspension, but also virtually abolished the [Ca²⁺]_iincrease. These observations may explain some of the inconsistencies among previous investigations of vWF-induced calcium signaling. Moreover, similar problems may arise in studies with other adhesive proteins.     

The canonical transient receptor potential 6 (TRPC6) channel is sensitive to extracellular pH in mouse platelets

The canonical transient receptor potential-6 (TRPC6) is a receptor-activated non-selective Ca^2 + channel regulated by a variety of modulators such as diacylglycerol, Ca^2 +/calmodulin or phosphorylation. The present study is aimed to investigate whether different situations, such as acidic pH, exposure to reactive oxygen species (ROS) or hypoxic-like conditions modulate TRPC6 channel function. Here we show normal aggregation and Ca^2 + mobilization stimulated by thrombin in TRPC6 KO platelets; however, OAG (1-oleoyl-2-acetyl-sn-glycerol)-evoked Ca^2 + entry was attenuated in the absence of TRPC6. Exposure of mouse platelets to acidic pH resulted in abolishment of thrombin-evoked aggregation and attenuated platelet aggregation induced by thapsigargin (TG) or OAG. Both OAG-induced Ca^2 + entry and platelet aggregation were greatly attenuated in cells expressing TRPC6 channels. Exposure of platelets to H₂O₂ or deferoxamine did not clearly alter thrombin, TG or OAG-induced platelet aggregation. Our results indicate that TRPC6 is sensitive to acidic pH but not to exposure to ROS or hypoxic-like conditions, which might be involved in the pathogenesis of the altered platelet responsiveness to DAG-generating agonists in disorders associated to acidic pH.     

Platelet membrane glycoprotein IIb heavy chain forms a complex with glycoprotein IIIa that binds Arg-Gly-Asp peptides

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-IIIa heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioiodinated platelets were treated with 1% 2-mercaptoethanol for 18 hours at 4 degrees C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipeptide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-IIIa complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-IIIa complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-IIIa complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-IIIa complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.