Familial macrothrombocytopenia associated with decreased glycosylation of platelet membrane glycoprotein IV

A case of hereditary thrombocytopenia with large platelets (familial macrothrombocytopenia, FM) is reported. Studies on the platelets from the propositus showed decreased glycosylation of platelet membrane glycoprotein IV, which would distinguish the case from other FM previously described.     

Dysfunctional platelet glycoprotein IIb/IIIa associated with a platelet release defect: A family study

We are reporting on a 36-year-old white female with a bleeding history attributed to dysfunctional platelet glycoprotein IIb/IIIa (GPIIb/IIIa) and a coexisting platelet release defect. Platelet aggregation studies (PAS) revealed markedly diminished to absent responses to ADP, epinephrine, collagen and arachidonic acid; the ristocetin response was normal. ATP content was normal with poor release to the agonists as measured by luminescent technique. DDAVP infusion shortened bleeding time from 13.5 min to 8.0 and 12 min (at 1 and 2 hours). Flow cytometry and immunoblotting revealed normal amounts of GPIIb and diminished GPIIIa (50% of control). Using a previously reported ELISA which measures the binding of GPIIb/IIIa to immobilized fibrinogen, the patient's platelet extract showed no binding to fibrinogen. Both the father and mother were found to have decreased PAS responses and normal amounts of GPIIb/IIIa determined by both Western blot and flow cytometry. However, the ELISA showed decreased binding of their GPIIb/IIIa to fibrinogen (71% and 62% as compared to controls, respectively). The patient's dysfunctional fibrinogen receptor was clearly demonstrated by the ELISA. The parents had moderately reduced GPIIb/IIIa function in this assay, but they did not demonstrate a reduced GPIIIa as was noted in the patient. The parents' PAS indicated a platelet release defect. These findings suggest an inherited platelet release defect and a dysfunctional GPIIIa. The partial response to DDAVP would be compatible with the presence of a platelet release defect. © 1993 Wiley-Liss, Inc.     

In vitro platelet adhesion to vascular subendothelium: studies in patients with polycythaemia vera

In this work we studied platelet adhesion to subendothelial surfaces in 10 patients with polycythaemia vera and 10 healthy volunteers at 40% Hct (corresponding to the mean value of our control group) and 55% Hct (a value roughly corresponding to the mean Hct in polycythaemic patients). Platelet concentration was kept constant at 2.0-2.5 X 10(11)/l. The results indicate that there was a statistically significant increase in adhesion both in controls and in patients with Hct varying from 40% to 55%. The contribution of the higher Hct in promoting platelet adhesion was comparable in the two groups. When red blood cells (RBC) from the patients were tested with platelets from healthy volunteers in cross-over experiments, they promoted adhesion in the same way as control RBC. Similarly, when patients' platelets were mixed with control RBC, adhesion was the same as control platelets. These data indicate that platelet and RBC contribution to this parameter are not significantly modified in this group of polycythaemic patients, provided that platelet and RBC values are adjusted to control range.     

Biosynthetic defect in platelet glycoprotein IX mutants associated with Bernard-Soulier syndrome

To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib- IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.     

Analysis of platelet adhesion to a collagen-coated surface under flow conditions: the involvement of glycoprotein VI in the platelet adhesion

Platelet adhesion to the exposed surface of the extracellular matrix in flowing blood is the first and critical reaction for in vivo thrombus formation. However, the mechanism of this in vivo platelet adhesion has yet to be studied extensively. One of the reasons for this is the lack of a practical assay method for assessing platelet adhesion under flow conditions. We have devised an assay method (the fluorescent adhesion assay) that is based on the technique originally reported by Hubbell and McIntire (Biomaterials 7:354, 1986) with some modifications to make it more amenable for assaying small samples and have developed an analysis method to quantify the extent of platelet adhesion and aggregation from fluorescence images by using a computer-assisted image analysis system. In our assay, platelet adhesion, expressed as the percentage of the area covered by adhered platelets, was found to increase biphasically as a function of time. In the first phase, platelets interacted with the coated collagen, transiently stopping on the surface; we called this reaction the temporary arrest. In the second phase, platelets adhered much more rapidly and permanently on the surface, and this adhesion was dependent on the shear rate; platelets formed aggregates in this phase. We used our assay to analyze the effects of platelet aggregation inhibitors on platelet adhesion. All three examined inhibitors, EDTA (10 mmol/L), antiglycoprotein (GP) IIb/IIIa, and GRGDS peptide (1 mmol/L), inhibited the second phase adhesion in flowing blood. Furthermore, GPVI-deficient platelets also showed defective second-phase adhesion under the same conditions. These results suggested that GPIIb/IIIa activation and GPVI contribute to the reaction inducing the second phase. The second-phase adhesion has been extensively investigated, and the consensus is that this reaction is mainly attributable to the platelet-platelet interaction. In this report, we were able to detect an earlier reaction, the temporary arrest. This temporary arrest would reflect the fast and weak interaction between platelet GPIb/IX and collagen-von Willebrand factor complexes on the collagen-coated surface.     

Effects of dipyridamole and low-dose aspirin therapy on platelet adhesion to vascular subendothelium

The effect on platelet function of low-dose aspirin (ASA) and dipyridamole alone or in combination was evaluated after repeated dosing in 5 healthy volunteers. The subjects were treated according to a randomized, single-blind, crossover design with 150 mg of dipyridamole, 25 mg of ASA, the 2 drugs together or placebo twice a day for 3 days. Platelet adhesion was evaluated using an experimental model of adhesion to rat aorta subendothelium under controlled hemodynamic conditions in the presence of red blood cells. Dipyridamole significantly reduced platelet adhesion both alone and in combination with ASA. ASA by itself did not significantly modify platelet adhesion, but completely blocked serum thromboxane production and platelet aggregation by arachidonic acid. Thus, low-dose ASA and dipyridamole may have a complementary action, modifying at the same time 2 platelet functions, adhesion and aggregation, both relevant in the pathogenesis of thrombosis.     

The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium

Platelet adherence to human artery subendothelium in blood from eight normal subjects, four patients with Glanzmann's thrombasthenia (deficiency of platelet membrane glycoproteins IIb and IIIa: GPIIb-IIIa), two patients with Bernard-Soulier syndrome (deficiency of platelet membrane glycoprotein Ib: GPIb) and one patient with von Willebrand's disease (VWD subtype III. deficient in factor VIII-von Willebrand factor: FVIII-VWF) was compared at various wall shear rates (300, 500, 1000, 1800 and 2500 s-1). Platelet adherence in blood from the patients with Glanzmann's thrombasthenia was within the normal range at shear rates below 1000 s-1. There was some decrease in adhesion at higher shear rates and platelets were less spread out on the subendothelium than normally at all shear rates. Platelet aggregate formation was almost totally absent. Platelet adherence in blood from patients with the Bernard-Soulier syndrome was strongly impaired at all shear rates. Platelet adherence in blood from the patient with VWD subtype III was normal at shear rates of 300 and 500 s-1, but impaired at shear rates above 1000 s-1. Aggregate formation was also decreased at these shear rates. Platelet adhesion was strongly inhibited by a monoclonal antibody against glycoprotein Ib, which had previously been shown to inhibit ristocetin-induced aggregation, at shear rates of 500 and 1800 s-1 but not at 300 s-1. Platelet adhesion at 1800 s-1 was also inhibited, though to a lesser extent, by two antibodies against GPIIb-IIIa. These antibodies also inhibited platelet aggregate formation. The data indicates that GPIb is involved in adhesion at the same shear rates as von Willebrand factor. Absence or inhibition of GPIIb-IIIa primarily causes a defect of aggregate formation but GPIIb-IIIa may also play a role in adhesion, particularly at high shear rates. The defect of adhesion in the Bernard-Soulier syndrome may be dependent on factors other than a deficiency of GPIb alone.     

Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder

Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.     

The human platelet membrane glycoprotein IIb/IIIa complex: a multi functional adhesion receptor.

The glycoprotein GPIIb/IIIa complex is a major constituent of the platelet membrane; it plays an important role in platelet adhesion and aggregation. The complex is a member of the integrin superfamily. Integrins are related membrane receptors which mediate the adhesive interactions of a variety of cells; they specifically recognize the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. The GPIIb/IIIa complex of activated platelets can bind fibrinogen, von Willebrand factor, fibronectin, vitronectin and thrombospondin. Platelets are activated by a variety of signals including extracellular matrix molecules and soluble factors; upon platelet activation the complex undergoes a conformational change, thus permitting the macromolecular ligands access to their binding sites. In turn, fibrinogen binding results in a receptor modification and neoantigens exposure; such events may participate in signal transduction. The adhesive proteins compete reciprocally for binding to GPIIb/IIIa, and the complex binds to different domains of them, thus creating multiple interactions with the ligands.     

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

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

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

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

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.     

Loss of blood platelet adhesion after heating native and cultured human subendothelium to 100 degrees Celcius

STUDY OBJECTIVE--Balloon angioplasty produces mechanical vessel wall injury that leads to substantial blood platelet deposition at the angioplasty site. The aim of the study was to determine whether thermal angioplasty might, by contrast, reduce platelet adhesion by denaturation of subendothelial adhesive proteins. DESIGN--Native and cultured human subendothelium was briefly heated to greater than or equal to 100 degrees C by laser irradiation or to 50-100 degrees C by immersion in preheated phosphate buffered saline (PBS). Subsequently, the subendothelium was exposed for 5 min to flowing human blood at a shear rate of 1300 s-1. Blood platelet adhesion to the subendothelium was determined quantitatively. EXPERIMENTAL MATERIAL--Human umbilical arteries were used and the subendothelial matrix derived from cultured umbilical vein endothelial cells. MEASUREMENTS AND RESULTS--After heating arterial subendothelium by laser irradiation to greater than or equal to 100 degrees C, zero platelet adhesion was found v 36(SD 2)% adhesion to the non-heated surface (p less than 0.001). After laser heating of the subendothelial matrix to greater than or equal to 100 degrees C, platelet adhesion was absent in 10/10 experiments (p less than 0.01). After heating the matrix to 100 degrees C by immersion in PBS, platelet adhesion was reduced to 5(5)% v 31(7)% at 37 degrees C (p less than 0.001). CONCLUSIONS--These in vitro results, if extrapolated to catheter interventions, suggest that thermal injury to the vessel wall by laser angioplasty or other thermal angioplasty methods may provide a basic and clinically relevant advantage over mechanical angioplasty modalities, because of a potentially reduced risk of complications related to platelet adhesion.     

Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system

The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3' end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.     

Morphological and morphometric characterization of platelet adhesion to the exposed subendothelium of the rabbit thoracic aorta in vivo

We have quantitated platelet adhesion to the exposed subendothelium of the balloon-deendothelialized rabbit descending thoracic aorta in vivo. Platelet adhesion was studied for elapsed times of 10 sec to 3 hr after injury. Platelet surface coverage of the thoracic aorta was measured by an intercept-counting light microscopy technique and a novel transmission electron microscopy technique. Both techniques gave similar results. Platelets adhered and spread on the exposed subendothelium forming a monolayer at all time intervals greater than 10 sec. However, platelets did not cover the entire surface. Coverage increased with time and reached a maximum of nearly 90% 3 hr after injury. Gaps between adhering platelets decreased with time and averaged approximately 1000 nm. No mural platelet thrombi were observed at any elapsed time. Comparison of our results with those reported by H. R. Baumgartner (1973, Microvasc. Res.5, 167–179) for the rabbit abdominal aorta in vivo suggests that platelet adhesion to the exposed subendothelium may be less extensive in the thoracic aorta.     

A platelet defect in a patient with eosinophilic leukaemia: absent ristocetin-induced platelet aggregation associated with a reduced platelet sialic acid content.

Platelets from a patient with eosinophilic leukaemia were not aggregated by ristocetin. The defect was not corrected by normal human plasma and was due to a platelet abnormality. The patient's platelets also showed a diminished sensitivity to aggregation by bovine factor VIIIVWF. The defect was not associated with a prolonged bleeding time. No abnormalities were detected in ADP, collagen or thrombin-induced platelet aggregation. Biochemical studies showed that the platelets were deficient in sialic acid. This deficiency was associated with a reduced staining for glycoprotein I following SDS-polyacrylamide gel electrophoresis. The results suggest an acquired platelet surface abnormality.     

Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.     

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.