Protein disulphide isomerase mediates platelet aggregation and secretion

Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti-PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti-PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC-1), the rabbit anti-PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Temperature dependence of plasmin-induced activation or inhibition of human platelets.

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.     

Platelet activation by sustained exposure to low-dose plasmin.

Plasmin has been reported to activate and inhibit platelet function depending on dose and exposure temperature. The present study examines the induction of fibrinogen-dependent platelet aggregation following prolonged (60 min) platelet exposure to very low doses of plasmin (0.05 CU/ml) at either 22 or 37 degrees C. Maximum aggregation [mean +/- SD, 60 +/- 19 light transmission units (LTU); n = 43] occurred following platelet exposure to plasmin at 22 degrees C, but significant platelet aggregation (28 +/- 4 LTU, n = 3) also occurred following plasmin treatment at 37 degrees C. Plasmin-induced platelet aggregates appeared microscopically larger than aggregates of adenosine diphosphate (ADP)-activated platelets, and were less reversible. Aggregated plasmin-treated platelets also expressed more procoagulant activity than platelets aggregated with ADP, as reflected by shortening of the plasma kaolin recalcification time. Aggregation of platelets exposed to very low doses of plasmin was not accompanied by dense or alpha-granule secretion, and was unaffected by ADP antagonists or aspirin. Partial inhibition of platelet aggregation, however, was achieved with metabolic inhibitors, PGE1, and inhibitors of phosphoinositide 3-kinase or protein kinase C. Although fibrinogen was required for plasmin-treated platelet aggregation, [125I]-fibrinogen binding comprised only 58 +/- 3% (n = 3) of fibrinogen binding associated with ADP aggregated platelets. This was consistent with observed decreases in reptilase-induced fibrin clot retraction. Taken together, these data suggest that sustained exposure of platelets to very low plasmin doses leads to platelet activation and thus may contribute to thrombotic complications in vivo.     

Synergistic action of two murine monoclonal antibodies that inhibit ADP- induced platelet aggregation without blocking fibrinogen binding

We have used two murine monoclonal antibodies, each directed against one component of the human platelet membrane glycoprotein (GP) IIb-IIIa complex, to examine further the molecular requirements for fibrinogen binding to the platelet surface and subsequent platelet-platelet cohesion (aggregation). Although neither AP3, which is directed against GPIIIa, nor Tab, which is specific for GPIIb, were individually able to inhibit adenosine diphosphate (ADP)-induced fibrinogen binding, platelet aggregation, or secretion, the combination of AP3 and Tab completely abolished platelet aggregation and the release reaction. Unexpectedly, this synergistic inhibition of platelet-platelet cohesion occurred in the presence of apparently normal fibrinogen binding. Both the number of fibrinogen molecules bound and the dissociation constant for fibrinogen binding remained essentially unchanged in the presence of these two antibodies. Inhibition of aggregation was dependent upon the divalency of both AP3 and Tab because substitution of Fab fragments of either antibody for the intact IgG resulted in a complete restoration of both aggregation and secretion. In contrast to ADP induction, thrombin-activated platelets neither aggregated nor bound fibrinogen in the presence of AP3 plus Tab but were fully capable of secretion, which illustrated the multiple mechanisms by which the platelet surface can respond to different agonists. These data demonstrate that fibrinogen binding to the platelet surface alone is not sufficient to support platelet-platelet cohesion and that an additional post-fibrinogen-binding event(s) that is inhibitable by these two monoclonal antibodies may be required.     

Structural changes in platelet glycoprotein IIb/IIIa by plasmin: determinants and functional consequences

Plasmin exposure modulates platelet aggregation responses, but a direct effect of plasmin on the platelet fibrinogen receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), has never been conclusively shown in a plasma milieu. To examine this issue, we incubated platelets in platelet-rich plasma with plasmin and measured the effect of this treatment on platelet aggregation, fibrinogen binding, and the structural integrity of GPIIb/IIIa. Plasmin treatment reduced maximal reversible fibrinogen binding in a dose-dependent fashion, and this reduction in binding was accompanied by a correlative reduction in the maximal rate of aggregation. Immunoblots performed with polyclonal antibodies against GPIIb/IIIa showed that GPIIIa had been cleaved by plasmin, but this cleavage was detected only after subsequent degradation of the solubilized GPIIb/IIIa with Staphylococcus aureus V8 (Glu-C) endoprotease. Peptide sequence analysis showed that cleavage occurred at the lys444-pro445 bond in the first cysteine-rich repeat domain of GPIIIa a unique proteolytic event observed only in the presence of plasma fibrinogen. These observations suggest that plasmin modifies GPIIIa by a unique proteolytic event in plasma that is dependent on fibrinogen binding and, consequently, is accompanied by significant reductions in fibrinogen binding and aggregation response.     

The mechanism of heparin-induced platelet aggregation

When heparin was added to platelet-rich plasma, mild but irreversible platelet aggregation was demonstrated. This platelet response was not accompanied by release of alpha-granules and dense body constituents, nor by prostaglandin biosynthesis. It did, however, require metabolic energy and divalent cations as metabolic inhibitors (anti-mycin A and 6-deoxyglucose) and EDTA blocked the reaction. Bernard-Soulier syndrome platelets, which lack glycoprotein (GP) Ib, but not Glanzmann's Thrombasthenia platelets, which lack GP IIb/IIIa, were aggregated by heparin. Monoclonal antibody (mAb) against GP IIb/IIIa, but not mAb against GP Ib, strongly inhibited the reaction. These combined results suggest the participation of GP IIb/IIIa but not GP Ib in heparin-induced platelet aggregation. Fibrinogen was a cofactor in the reaction as gel-filtered platelets were unreactive to heparin but addition of fibrinogen restored their reactivity. Antithrombin III and fibronectin inhibited platelet response to heparin, suggesting that these proteins may protect platelets from aggregation by heparin.     

The secretory release reaction initiated by complement proteins C5b-9 occurs without platelet aggregation through glycoprotein IIb-IIIa

The secretory and aggregation responses of stirred platelets exposed to complement proteins C5b-9 was investigated. C5b-9 assembly on the platelet surface resulted in the release of dense granule adenosine triphosphate (ATP) accompanied by a decrease in sample turbidity, but no detectable cell lysis. Inhibition of cellular protein kinase C completely blocked the C5b-9 initiated release of ATP, confirming the secretory nature of this response. Addition of fibrinogen (up to 1 mg/mL) had no effect on either the release of ATP or the decreased turbidity observed for C5b-9 cells. Addition of the peptides Arg-Gly- Asp-Ser (RGDS) and fibrinogen gamma-chain carboxyl-terminal (gamma 397- 411) at concentrations sufficient to completely block fibrinogen binding to GP IIb-IIIa had no effect on either C5b-9 induced dense granule secretion or the associated turbidity change. Microscopic examination and electronic particle counting of the stirred platelet suspensions confirmed that no aggregation of C5b-9 platelets occurred even when these cells were stirred in the presence of fibrinogen. The capacity of the C5b-9 proteins to initiate platelet secretion without activation of cell surface glycoprotein (GP) IIb-IIIa suggests a mechanism for intravascular dissemination of activated platelets during complement activation in vivo.     

Plasmin inhibition of thrombin-induced platelet aggregation.

The effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10-20 muM, PLASMIN (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin's inhibition of aggregation. Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction. Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin-treated platelets, upon centrifugation and treatment with SBTI. With the use of a "cold initiation" technique, the release by thrombin of 46.7 plus or minus 6.7 (mean plus or minus SEM) mu-g of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by STBI) aggregated normally upon addition of as little as 10 mu-g human plasma fibrinogen per mg platelet protein. It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.     

Recent development of peptides from glycoproteins IIb (alphaIIb) and IIIa (beta3) that inhibit platelet fibrinogen binding

The glycoprotein (GP) IIb/IIIa (alphaIIbbeta3) found on platelets binds fibrinogen when platelets are activated, thereby mediating the platelet aggregation process. Blockading of alphaIIbbeta3 has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. This inhibition of platelet aggregation is thought to be an effective therapeutic approach to various thromboembolic syndromes. The development of various forms of alphalambdapietaalpha;IIbbeta3 inhibitors has resulted in the inhibition of platelet aggregation, although studies of alphaIIbbeta3 receptor function and various alphaIIbbeta3 inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having non-platelet effects. This review describes the newly derived peptides from 1) glycoprotein IIb (alphaIIb) that interferes with platelet aggregation by inhibiting the binding of fibrinogen to alphaIIbbeta3 and from 2) GP IIIa (beta3) by blocking the alphaIIbbeta3 complex formation. These peptides may become effective agents to block the interaction of ADP, type I collagen, and type III collagen (type I collagen and type III collagen are present in abundant amounts in blood vessel walls) with platelets.     

Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.     

Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.     

Decreased accessibility of platelet-bound fibrinogen to antibody and enzyme probes

The binding of fibrinogen to platelets is a multiphasic process leading to apparently nonreversible associations between fibrinogen and stimulated platelets. To further investigate changes in platelet- fibrinogen interactions, the present study examined the accessibility of platelet-bound fibrinogen and its GPIIb-IIIa receptor to antibody and enzyme probes as a function of time after platelet stimulation with adenosine diphosphate (ADP). Whereas only minimal changes in fibrinogen and 10E5 binding were observed within 60 minutes after platelet stimulation and equilibrium fibrinogen binding, the binding of polyclonal antifibrinogen antibodies decreased significantly (75% +/- 13%, mean +/- SD, n = 9). Similar decreases were noted with rabbit antifibrinogen Fab and F(ab')2 fragments. In addition, plasmin (32 mU/mL) added to platelets five minutes compared with 60 minutes after equilibrium fibrinogen binding dissociated 52% +/- 12% compared with 33% +/- 7% of platelet-bound fibrinogen in five minutes, and 83% +/- 15% compared with 66% +/- 14% of bound fibrinogen in 15 minutes. No difference in plasmin cleavage products was observed, however, by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). Complete fibrinogen dissociation occurred 30 minutes after plasmin addition, confirming that fibrinogen was not internalized. In contrast, dissociation of platelet-bound fibrinogen by chymotrypsin was less affected by time after equilibrium fibrinogen binding, and minimal changes in antifibrinogen antibody recognition and plasmin-induced dissociation of fibrinogen bound to stimulated but glutaraldehyde-fixed platelets were observed. The data suggest that ADP-induced fibrinogen binding to fresh platelets is accompanied by progressive rearrangements of fibrinogen on the platelet surface.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

In vitro effect of plasmin on human platelet function in plasma. Inhibition of aggregation caused by fibrinogenolysis.

BACKGROUND. Plasmin has been reported both to activate platelets and to inhibit platelet functions. The latter effect was thought to be caused by proteolysis of the main membrane glycoproteins. METHODS AND RESULTS. We found that incubation of citrated human platelet-rich plasma with streptokinase (SK) (300 IU/ml) does not produce any detectable activation but leads to a time-dependent inhibition of ADP-induced aggregation accompanied by substantial fibrinogenolysis. These effects were abrogated by previous addition of a plasmin inhibitor, aprotinin. Crossover experiments (SK-treated or control platelets mixed with SK-treated or control plasma) demonstrated that the platelets remained functional and that the aggregation defect was caused by fibrinogenolysis. Further experiments (addition of purified fibrinogen to fibrinogen-depleted plasma with either SK or thrombin) suggested that in addition to the low residual level of fibrinogen, fibrinogen degradation products had an inhibitory effect. Under the same conditions, tissue-type plasminogen activator (t-PA) (3,000 ng/ml) had no effect on platelet aggregation, and plasma fibrinogen was not significantly lowered. The effects on glycoproteins IIb-IIIa of incubation with SK, t-PA, or plasmin were assessed with immunoblots with murine monoclonal antibodies directed against either part of the complex, which is the receptor for fibrinogen. Proteolysis was detected only in the presence of EDTA, a potent chelator of divalent cations. CONCLUSIONS. The incubation of human platelets in citrated plasma with SK concentrations obtained during therapy leads to an aggregation defect that is related to the decrease in fibrinogen, the adhesive protein involved in this function, and to the impeding effect of fibrinogen degradation products on its binding onto platelets but not to an alteration of the corresponding platelet receptor, the heterodimer glycoproteins IIb-IIIa.     

Interaction of a Monoclonal Antibody to Glycoprotein IV (CD36) with Human Platelets and its Effect on Platelet Function

FA6-152, a monoclonal antibody to platelet membrane glycoprotein IV (CP IV), was used to quantify the expression of this glycoprotein on platelets, as well as to evaluate its role in platelet aggregation. On resting platelets, 19 400 ± 7700 molecules of the (125)I-labelled IgC could bind per platelet (n = 20). Binding was not modified following stimulation of the platelets with ADP (10 μmol/l) or thrombin (0.1 U/ml). Fab fragments prepared from the antibody by papain digestion also bound to the platelet surface in a saturable manner. Both the intact IgC and its Fab fragments were found to inhibit platelet aggregation and secretion induced by ADP or collagen in platelet-rich plasma and by thrombin in platelet suspensions. Under nonstirred conditions, whereby the release reaction was only minimally affected, the antibody markedly inhibited thrombin-induced surface expression of α-granule thrombospondin (TSP), whereas it did not alter the concomitant expression of α-granule fibrinogen. In addition, electron microscopy revealed a predominant distribution of TSP and T;P IV on pseudopodia and between adherent cells on thrombin-stimulated platelets. These findings thus support the hypothesis that the interaction of TSP with GP IV on the platelet surface is required for an optimal platelet aggregation/secretion process to occur.     

Internalization of bound fibrinogen modulates platelet aggregation

In agonist-stimulated platelets, the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) is converted from an inactive to an active fibrinogen receptor, thereby mediating platelet aggregation. With time after agonist addition, at least two events occur: fibrinogen becomes irreversibly bound to the platelet and, when stirring is delayed, platelets lose the ability to aggregate despite the presence of maximally bound fibrinogen. Because we previously identified an actively internalized pool of alpha IIb, beta 3 in platelets, we explored the possibility that both of these events might result from the internalization of fibrinogen bound to active alpha IIb beta 3. Under conditions of irreversible fibrinogen binding, fluorescence microscopy showed that biotinylated fibrinogen is rapidly internalized by activated platelets to a surface-inaccessible, intracellular pool. Flow cytometric analysis showed that the observed loss in accessibility to extracellular probes immediately precedes a loss in ability to the platelets to aggregate. Moreover, prevention of irreversible fibrinogen binding results in a prevention of internalization and a retention of aggregation capacity. Thus, the internalization of fibrinogen from the activated platelet surface appears to contribute not only to the irreversible phase of fibrinogen binding, but also to the downregulation of platelet adhesiveness. Fibrinogen internalization is therefore likely to represent a fundamental regulatory mechanism that modulates platelet function.     

Platelet interaction with subendothelial extracellular matrix: platelet- fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction

Cultured endothelial cells produce an extracellular matrix (ECM) to which platelets adhere and spread, ultimately resulting in platelet aggregation, thromboxane B2 production, and serotonin release. We have investigated the role of fibrinogen binding to the platelet GPIIb/IIIa complex in these reactions by comparing normal platelet-rich plasma (PRP), PRP from patients with Glanzman's thrombasthenia (whose platelets lack the GPIIb/IIIa complex), PRP in the presence of a monoclonal antibody that blocks the binding of fibrinogen to the GPIIb/IIIa complex, platelets washed free of fibrinogen, and washed platelets to which fibrinogen was added. Although platelet aggregation was virtually completely inhibited in the samples in which the normal interaction between fibrinogen and GPIIb/IIIa was impaired, adhesion of platelets to the matrix, spreading, and release of [14C]-serotonin were not affected. All of the platelet preparations released significant amounts of T X B2 with time, but there was a decrease in the amount produced by both the thrombasthenic and antibody-treated platelets. We conclude that the interaction of fibrinogen with platelet GPIIb/IIIa is not required for platelet adhesion to ECM or for adhesion-induced shape change or serotonin release. On the other hand, the platelet-fibrinogen interaction may play some role in augmenting adhesion-induced T X B2 production, and it is absolutely required for adhesion-induced platelet aggregation.     

Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot-reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti-glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non-stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.     

The Anti-aggregating Peptide KRDS Impairs a-granule Release, Whereas RGDS Does Not

The effects on platelet activation of two different tetrapeptides, KRDS present on human lactotransferrin and RGDS present on adhesive proteins such as human fibrinogen α-chain, were compared by a combination of morphological and functional techniques. Ultrastructural observations of α-thrombin stimulated platelets (0.05 U/ml), show strong platelet aggregation and full α-granule release. In the presence of RGDS (0.1-1 mM) aggregation was impaired but secretion was not blocked and platelets had released their α-granule contents. Platelets appeared uniformly degranulated with a dense central meshwork of microfilaments. In the presence of KRDS (0.5-1 mM), the platelets were activated with shape change and pseudopod formation. Aggregation was also impaired, but to a lesser extent since RGDS is active at a concentration as low as 0.1 mM, and, in contrast to RGDS, secretion was severely reduced. Electron microscopy showed that numerous α-granules were still scattered in the cytoplasmic matrix or often gathered in the centre of the platelet, but the majority of the open canalicular system cisternae remained clear. An immunoelectron microscopic study using immunogold and monospecific antibodies directed against fibrinogen and the a-granule membrane protein P-selectin (GMP 140) was performed. In the presence of RGDS, fibrinogen was released and P-selectin was translocated to the platelet surface; in contrast, in the presence of KRDS, fibrinogen remained localized in the α-granule, and the P-selectin associated with the a-granule. These observations were accompanied by some functional results: thrombin-induced platelet aggregation was inhibited by both peptides, and in contrast to RGDS, secretion was severely reduced in the presence of KRDS: serotonin release from dense granule was reduced by 73% compared to the control. These results show that these two tetrapeptides, in spite of some structural similarities, act differently in impairing platelet function. KRDS interfering with both the dense and α-granule release reaction may be a useful tool for a better understanding of the platelet secretion mechanism.