Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.     

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.     

Platelet receptors for collagens

Collagens belong to the constituents that determine the thrombogenicity of the vessel wall. Seven genetically distinct collagens-type I, III, IV, V, VI, VIII and XIII-have been identified in the vessel wall. The interaction of platelets with collagens is a complex process since collagens are not only potent platelet agonists but also adhesive proteins. In recent years several platelet membrane glycoproteins have been shown to be involved in platelet-collagen interactions. The mechanisms of platelet-collagen interaction can be divided into primary, direct interactions and secondary, indirect interactions. A number of direct receptors for the collagens have been proposed. The glycoprotein complex Ia/IIa, also called VLA(2), α(2)β(1) integrin or CD49b/CD29, meets several criteria as the major platelet receptor for different types of collagens. There is some evidence that glycoprotein IIIb, also called glycoprotein IV or CD36, functions at the earliest stages of collagen adhesion as a platelet collagen receptor. CD36 seems to be essential for type V collagen-induced platelet aggregation and adhesion. Another putative platelet-collagen receptor is P62, a glycoprotein with a molecular weight of 62 kDa under reducing conditions.     

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.     

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.     

Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.     

In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion

BACKGROUND AND OBJECTIVES: Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. DESIGN AND METHODS: The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. RESULTS: Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. INTERPRETATION AND CONCLUSIONS: Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.     

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.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets. Immunoprecipitation analyses indicate that CIB and glycoprotein IIb/IIIa (GPIIb/IIIa) interact with each other as platelets adhere to immobilized Fg, and together they associate with the platelet cytoskeleton. Introduction of anti-CIB antibody or GPIIb cytoplasmic peptide into platelets blocks lamellipodia but not filopodia formation. GPIIb peptide-induced inhibition of platelet spreading is recovered by the incorporation of recombinant CIB protein, suggesting that interaction between CIB and GPIIb/IIIa is required for progression from filopodial to spread morphologies. Further, anti-CIB- or GPIIb peptide-induced inhibition of platelet spreading can be overcome by the addition of exogenous adenosine diphosphate (ADP). These data suggest that formation of the CIB-GPIIb/IIIa complex may be necessary for initiation of downstream signaling events, such as ADP secretion, that lead to platelet spreading.     

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.     

Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (alpha 2 beta 1-integrin)

Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.     

Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.     

P-selectin expression, but not GPIIb/IIIa activation, is enhanced in the inflammatory stage of Takayasu's arteritis.

BACKGROUND: Inflammation and thrombosis are closely related processes, but the association between disease activity and thrombogenicity in Takayasu's arteritis (TA) is poorly understood. To investigate the link between platelet activation and disease activity, flow cytometric analyses of platelet P-selectin and activated GPIIb/IIIa expression were performed in patients with TA. METHODS AND RESULTS: Twenty-two patients with TA, classified into active (Group A, n = 9) and inactive (Group I, n = 13) according to blood-derived inflammatory markers, and 14 healthy age- and gender-matched controls (Group C) were studied. Compared with Group C, the mean fluorescence intensity of P-selectin in response to 0.1-10 micromol/L of ADP was significantly upregulated in Group A, but not in Group I. No differences in platelet GPIIb/IIIa expression in stimulated platelets were seen among the 3 groups. Standard platelet aggregation studies revealed that disease activity did not influence platelet aggregation by ADP. CONCLUSIONS: P-selectin expression, but not activated GPIIb/IIIa, is enhanced in ADP-activated platelets from patients in the inflammatory stage of TA. P-selectin may play a significant role in the inflammatory and thrombotic responses associated with intractable TA, presumably by inducing platelet-leukocyte interactions.     

MK-383 (Tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin α_IIb β₃ (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10^-7 mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

Development of peptide inhibitors to block type I and type III collagen-platelet interaction

Platelet-collagen interaction plays an important role in hemostasis and pathological thrombosis. Upon an injury to the subendothelium of a blood vessel wall, platelets attach to the denuded substrate, aggregate, and release biological substances. Many investigators are trying to explore blocking agents to interrupt the adhesion of platelets to the damaged walls. To this date, extensive studies on developing inhibitors for platelet glycoprotein IIb/IIIa have been conducted. There are many trial reports showing that the efficacy of these antagonists has not completely blocked the adhesion of platelets to the distal site(s) of the injury. Type I and type III collagen are present in abundant amounts in blood vessel walls and other laboratories have obtained various active peptides from collagen molecules to block type I and type III collagen-platelet interaction. We have tried to develop active peptides from platelet receptors for types I and III collagen to aberrant both type I and type III collagen-induced platelet aggregation. We have defined effective and specific peptide inhibitors for each type of collagen to block binding to washed platelets, platelet aggregation. and adhesion of washed platelets to rabbit aortic segments.     

Platelets deficient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V

The aggregation of platelets induced by collagens is considered an important step in primary hemostasis. Glycoprotein (GP) IIIb (GPIIIb, GPIV, CD36) has been proposed as a blood platelet receptor for collagen. Platelets from three healthy blood donors were shown to be clearly deficient in GPIIIb. These platelets aggregated normally in response to type I and III collagens. In addition, platelet factor 4, beta-thromboglobulin, and adenosine triphosphate (ATP) secretion in response to type I and III collagens was normal. The findings indicate that GPIIIb is not the major, essential collagen receptor for type I and III collagens. This would explain why all individuals with GPIIIb- deficient platelets examined so far are healthy and, in particular, show no apparent evidence of hemostatic problems. However, in contrast to control platelets, no aggregation and impaired platelet factor 4, beta-thromboglobulin, and ATP secretion was observed in response to type V collagen. Therefore, it is postulated that for type V collagen- induced aggregation both GPIa/IIa and GPIIIb are essential.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

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.     

Mechanisms underlying the inhibitory effects of lipopolysaccharide on human platelet adhesion

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 × 10⁸ platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01–300 µg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 µg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca²⁺ platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca²⁺, independently of cGMP generation and activation of GPIIb/IIIa complex.