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P Nurden

Publications and source records attributed to P Nurden.

At least 55 records · Page 3Linked to original sources
PubMed1997

Ultrastructural analysis of the distribution of the vitronectin receptor (alpha v beta 3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the alpha-granule membrane.

The vitronectin receptor (VnR or alpha v beta 3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the beta 3 subunit in common: GP IIb-IIIa complexes (or alpha IIb beta 3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the alpha v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, alpha v beta 3 was mostly detected in internal membrane systems including those of alpha-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of alpha v beta 3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb-IIIa, although intracellular staining was present in EM and alpha-granules were again labelled.

Blood Platelets↗
PubMed1997

Incidence of anti-mouse antibodies in thrombocytopenic patients with autoimmune disorders.

Idiopathic thrombotycopenic purpura (ITP) is an autoimmune disorder in which circulating autoantibodies react with target antigens on the platelet membrane. In order to identify the autoimmune response in ITP, two MAIPA (Monoclonal Antibody (MAb) Immobilization of Platelet Antigen) assays (MAIPA I and MAIPA II) were performed on sera from thrombocytopenic patients. In the classic MAIPA assay (MAIPA I), control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein. In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A positive MAIPA I test but a negative MAIPA II has been shown to result from the presence of serum antibodies recognizing mouse MAb to platelet glycoproteins used in the assay. We compared the frequency of such 'anti-mouse' antibodies in patients with thrombocytopenia associated or not with other autoimmune states and in healthy donors with a normal platelet count. Statistically significant differences were found in the incidence of anti-mouse antibodies between patients and healthy donors. Furthermore, the identity of the targeted mouse MAbs varied in sera from the patients. The detected anti-mouse antibodies may include anti-idiotypic antibodies produced against cross-reactive idiotypes shared by human and mouse anti-platelet antibodies.

Adolescent↗
PubMed1996

Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6.

The sequence beta (3)203-228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211-221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding-site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin-stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin-induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.

Adenosine Diphosphate↗
PubMed1996

The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system.

In this study, we show that the platelet surface expression of glycoprotein (GP) V is regulated by two independent mechanisms. While confirming that both thrombin and neutrophil elastase proteolyse GPV, we show that neutrophil cathepsin G, thrombin receptor activating peptide (TRAP), and a combination of ADP and epinephrine can each result in a decrease in the platelet surface expression of GPV by a nonproteolytic mechanism: a cytoskeletal-mediated redistribution of platelet surface GPV to the surface-connected canalicular system (SCCS). Four independent lines of evidence documented the nonproteolytic nature of this decrease in the platelet surface expression of GPV. First, flow cytometric studies showed that cathepsin G, TRAP, and ADP/epinephrine decreased the platelet surface expression of GPV without changing the total platelet content of GPV. Second, immunoelectron microscopy directly demonstrated translocation of GPV from the platelet surface to the SCCS. Third, the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV were fully reversible. Fourth, cytochalasin B, an inhibitor of actin polymerization, completely inhibited the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV. The cytoskeletal-mediated redistribution of GPV occurred in a whole blood milieu and at physiologic temperatures (37 degrees C) and extracellular calcium concentrations (2 mmol/L). This study also defines the diverse effects on GPV, GPIb, and GPIX of multiple important platelet agonists. Cathepsin G proteolysed platelet surface GPIb alpha, but redistributed platelet surface GPIX and GPV to the SCCS. Thrombin proteolysed platelet surface GPV, but redistributed platelet surface GPIb and GPIX to the SCCS. Both TRAP and ADP/epinephrine redistributed platelet surface GPIb, GPIX, and GPV to the SCCS. Elastase proteolysed platelet surface GPIb alpha and GPV, but, unlike the other agonists tested, neither proteolysed nor redistributed platelet surface GPIX. The experiments with TRAP showed that activation of the seven-transmembrane domain thrombin receptor can result in translocation of GPIb, GPIX, and GPV to the SCCS independently of the GPIb-mediated pathway of thrombin-induced platelet activation. This study also provides two additional lines of support for the recent report that GPV is noncovalently complexed with GPIb and GPIX in the platelet surface membrane. First, although only the GPIb alpha subunit of this putative complex is known to be directly linked to the platelet cytoskeleton via actin-binding protein, cytochalasin B inhibited the ADP/epinephrine-, cathepsin G-, and TRAP-induced decrease in platelet surface GPV. Second, triple labeling flow cytometric experiments showed that, on each individual platelet, the ADP/epinephrine-induced decrease and subsequent return of the platelet surface expression of GPV occurred simultaneously with the decrease and subsequent return of the platelet surface expression of GPIb. In summary, the platelet surface expression of GPV is regulated by two independent mechanisms: proteolysis and a reversible, cytoskeletal-mediated redistribution to the SCCS.

Actins↗
PubMed1996

Platelet activation and thrombosis: studies in a patient with essential thrombocythemia.

Recent advances permit the detection of activated platelets using specific monoclonal antibodies and flow cytometry. Nevertheless, there are few reports in which activated platelets have been studied over a period of time in patients at risk for thrombosis. Our patient S.D. has essential thrombocythemia and a prothrombotic state manifested in two major thrombotic episodes involving the portal vein and a mesenteric artery. Investigation revealed both spontaneous aggregation and hyperaggregability in response to ADP and the presence of activated platelets in platelet-rich plasma as revealed by flow cytometry. Interestingly, the activated platelets were recognized by an anti-RIBS ("receptor-induced binding site") monoclonal antibody that recognized bound fibrinogen but not by antibodies reactive with antigens whose presence on the platelet surface was secretion dependent. Treatment with aspirin inhibited spontaneous platelet aggregation but had little effect on the activated platelet profile. A change of therapy to ticlopidine suppressed expression of platelet activation markers. Treatment with ticlopidine has continued for 1 year so far without further thrombotic complications.

Adenosine Diphosphate↗
PubMed1996

Autoantibodies and anti-mouse antibodies in thrombocytopenic patients as assessed by different MAIPA assays.

Two MAIPA (monoclonal antibody [MAb] immobilization of platelet antigen) assays were performed to determine (a) autoantibodies to platelet glycoproteins (GP) and (b) serum antibodies recognizing mouse MAbs used in the assay. In MAIPA I, control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein (GP IIb-IIIa, Ib-IX, Ia-IIa, IV and p24). In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A series of 25 patients with autoimmune thrombocytopenic purpura (ATP) associated or not with other autoimmune states were examined. Autoantibodies (both MAIPA I and MAIPA II positive) or anti-mouse Abs (MAIPA I positive and MAIPA II negative) were frequent in both groups of patients. Statistically significant differences existed in the incidence of anti-mouse Abs between patients (56.5%) and healthy donors (10%). This suggests that their production may be related to thrombocytopenias associated with autoimmune disease. We speculate that the presence of anti-mouse antibodies could reflect an abnormality in the immunological modulation of the idiotypic network.

Adult↗
PubMed1996

Visualization of activation-dependent epitopes on glycoprotein IIb-IIIa complexes of platelets stimulated by thrombin: immunogold staining of ultrathin sections.

An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and immunogold staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but immunogold staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.

Antigens, Human Platelet↗
PubMed1996

Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation.

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

Adenosine Diphosphate↗
PubMed1996

Giant platelets, megakaryocytes and the expression of glycoprotein Ib-IX complexes.

The role of GP Ib-IX complexes in platelet adhesion was discovered from studies on patients with the Bernard-Soulier Syndrome (BSS). In this inherited disorder, the bulk of the platelets are round and giant. Furthermore, the platelet count is decreased, often severely so. The relationship between these abnormalities and the deficiency of GP Ib-IX is not well understood. In normal discoid platelets, the bulk of GP Ib-IX is found on the plasma membrane. After platelet activation by thrombin, this distribution changes and the majority of GP Ib-IX complexes are located within the surface-connected canalicular system (SCCS). The platelets now possess pseudopods and are spheroid. Cytoskeletal modifications accompany these changes. We now report that platelets of a BSS variant with a qualitative defect of GP Ib show no translocation of GP Ib in response to thrombin, suggesting that the linkage with the cytoskeleton is impaired. Morphological studies of megakaryocytes (MK) from BSS patients show an altered maturation and an abnormal development of the membrane systems, implying a role for GP Ib-IX in megakaryocytopoiesis. In a case of Epstein syndrome, where giant platelets and thrombocytopenia are associated with deafness and renal dysfunction, platelets possess the bulk of GP Ib-IX inside the SCCS but without signs of platelet activation as assessed by P-selectin expression. This patient also shows an impaired megakaryocytopoiesis and an irregular development of the demarcation membranes within the MK. As collagen IV mutations are a feature of the related Alport syndrome, we hypothesize that defects in the link between extracellular matrix proteins, membrane receptors and the cytoskeleton could be a common cause of giant platelet syndromes.

Bernard-Soulier Syndrome↗
PubMed1996

[A study of platelet activation following coronary angioplasty. Effect on acute occlusion and restenosis].

The development of monoclonal anti-platelet antibodies to activation-dependent epitopes has made possible the study of the appearance of activated platelets in the blood by cytometry. This was undertaken in 35 patients undergoing coronary angioplasty. Blood was sampled through the femoral venous catheter introducer before the procedure, at 3 minutes and 3, 24 and 48 hours after angioplasty. Systematic coronary angiography was performed at about 6 months and vessel diameters were quantified by a digitised technique. In general, platelet activation was not observed before angioplasty or in the first hours following the procedure. On the other hand, a significant number of activated platelets was observed secondarily : 7 +/- 7.3% at the 24th hour and 6.8 +/- 5.9% at the 48th hour. There was a significant correlation between the level of platelet activation before angioplasty and that at the 24th hour (r = 0.52, p = 0.01). There was a tendency to more activated circulating platelets before and 24 hours after angioplasty in patients with acute occlusion. The correlation coefficient between the level of activated platelets 24 hours after angioplasty and progression of stenosis at 6 months was not statistically significant (r = 0.29). Therefore, the authors demonstrated platelet activation, which was sometimes considerable, inconstantly in circulating venous blood of patients with angina 24 hours after coronary angioplasty despite treatment with aspirin. Activated platelets may play a role in constituting acute occlusion by thrombosis but their detection was not predictive of restenosis.

Adult↗
PubMed1996

PAICA: a method for characterizing platelet-associated antibodies--its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3.

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA ("Platelet-Associated IgG Characterization Assay"), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient's own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA ("Monoclonal Antibody-specific Immobilization of Platelet Antigens"). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

Adult↗
PubMed1995

A patient with autoimmune thrombocytopenic purpura demonstrating serum antibodies reactive with mouse cross-reactive idiotypes.

Autoimmune thrombocytopenic purpura (ATP) is a syndrome of destructive thrombocytopenia due to platelet-binding antibodies. We report the case of a young woman (C.V.) who has a history of chronic ATP with severe but transient bouts of thrombocytopenia. Using the classic monoclonal antibody (MAb) immobilization of platelet antigens (MAIPA) assay to screen serum antibody specificity, results were strongly positive with MAbs to glycoproteins (GPs) Ib-IX, Ia-IIa, IV, and p24, but weakly positive or negative for GP IIb-IIIa. In contrast, a two-step incubation assay (MAIPA II), in which platelets were incubated sequentially with C.V. serum and the murine MAb, gave negative results for all GPs. Affinity chromatography performed using Bx-1, a MAb to GP Ib, showed that the patient's serum contained antibodies to determinants expressed by mouse immunoglobulins. These were present on Fab fragments on Bx-1. A survey of sera from other patients with thrombocytopenia of immune origin revealed that antibodies reactive with selected idiotypes of mouse MAbs were not infrequent and raises the question of their role in the thrombocytopenia.

Adult↗
PubMed1995

Flow cytometry reveals activated GP IIb-IIIa complexes on platelets from patients undergoing thrombolytic therapy after acute myocardial infarction.

We report the detection of activated GP IIb-IIIa complexes on platelets of patients undergoing thrombolytic therapy after acute myocardial infarction. Protocols were established for the monoclonal antibodies (mAbs): VH10, anti-P-selectin, a marker of platelet secretion; 9F9 and F26, two anti-RIBS (receptor-induced binding sites) mAbs specific for fibrinogen (Fg) bound to the GP IIb-IIIa receptor. Of ten patients studied: two were treated with streptokinase, four with APSAC (anisoylated plasminogen-streptokinase activator complex), and three with rt-PA. Platelets were tested on at least five occasions in the week following therapy. The percentage of platelets positive with 9F9 was often high, and reached a maximum within three days. By this time, plasma Fg levels, which fell during fibrinolysis, had begun to return to normal. Levels of activated platelets had fallen to baseline after 7 days. PAC-1, a mAb which binds directly to the activated GP IIb-IIIa complex, confirmed the results with 9F9, but F26 was a less sensitive probe. Binding of the anti-P-selectin mAb (VH10) was low, showing that little secretion had occurred. A concentration-dependent inhibition of 9F9 binding by RGDW peptide, a competitive inhibitor for Fg on GP IIb-IIIa, confirmed that Fg (or epitope-containing degradation products) were being located by the antibody. The activation of GP IIb-IIIa occurred despite the patients receiving aspirin and heparin. Thus platelets of some fibrinolytic patients have an increased tendency for surface activation within the first 72 h after treatment, a finding which would be compatible with an increased thrombotic tendency.

Adult↗
PubMed1995

Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide.

In 1990 we reported that GP Ib-IX complexes accumulated within the surface-connected canalicular system (SCCS) of thrombin-stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Ib alpha in electron microscopy with immunogold staining performed on ultrathin sections of resin-embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti-GP Ib-IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either alpha-thrombin or TRAP-14-mer peptide. In all cases a decreased density of GP Ib-IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP-14-mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib-IX complexes which follow thrombin-induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP-14-mer peptide.

Blood Platelets↗
PubMed1995

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

Adenosine Diphosphate↗
PubMed1994

Markers of platelet activation in coronary heart disease patients.

We have applied flow cytometry to the detection of activated platelets in patients with coronary heart disease. Paraformaldehyde-fixed platelets were incubated with one of the following monoclonal antibodies (MAbs): Bx-1 (anti-GP Ib), AP-2 (anti-GP IIb-IIIa complex), VH10 (anti-GMP-140, a glycoprotein of the alpha-granule membrane), or PAC-1 (directed against an activation-dependent determinant on GP IIb-IIIa complexes). Bound antibody was quantitated after the addition of FITC-conjugated anti-immunoglobulin. This report highlights studies on 16 unstable angina patients undergoing transluminal angioplasty. Blood samples were taken at different periods before and after the angioplasty. Levels of activated platelets were variable, remaining in the 2-4% range of control donors for some, but increasing to 10-30% post-angioplasty for others (despite all patients receiving heparin and aspirin). Maximum numbers of activated platelets were detected at 24 or 48 h. Nonetheless, the amount of antibody bound to individual platelets rarely reached the levels seen when control platelets were stimulated with thrombin in vitro. Results with VH10 and PAC-1 often, but not always, correlated suggesting different pathways of platelet activation.

Angina, Unstable↗
PubMed1994

Glycoprotein Ia-IIa (VLA-2) and glycoprotein Ib-IX complexes are processed independently during thrombin-induced platelet activation.

We have previously shown that when human platelets are stimulated by thrombin, glycoprotein Ib-IX (GP Ib-IX) complexes are cleared to the surface-connected canalicular system (Blood 1990;76:1503). The question arose as to whether GP Ia-IIa complexes (VLA-2), another adhesion receptor thought to be linked to the membrane cytoskeleton, behaved similarly. Monoclonal antibodies to GP Ia-IIa were used in either (1) immunofluorescence procedures and flow cytometry or (2) immunogold staining and electron microscopy. In flow cytometry, VLA-2 was shown to have a low but variable expression in the platelets of different donors. Immunogold staining showed that the complexes were regularly distributed over the platelet surface. This was best seen after staining of paraformaldehyde-fixed whole mounts, where bound antibodies were often visualized in small clusters. The surface expression of VLA-2 receptors increased somewhat after thrombin stimulation, the receptors coming from a small intracellular pool revealed by flow cytometric analysis of Triton X-100-permeabilized cells. Immunogold staining showed that after activation the receptors were equally as present on pseudopods as on the peripheral zone of the platelet. Down-regulation, as seen with GP Ib-IX complexes, was not observed. Our results therefore show that two-way trafficking of adhesion receptors can occur during platelet activation. This would imply that GP Ib-IX and VLA-2 are attached to different elements within the membrane cytoskeleton.

Blood Platelets↗