Inheritance of the human platelet alloantigen, PlA1, in type I Glanzmann''s thrombasthenia.

The hereditary of the human platelet alloantigen, PlA1, has been studied in Glanzmann's thrombasthenia. The PlA1 content of platelets from three patients, 20 kindred of these patients, including parents and siblings, and 15 unrelated normal individuals was determined using immunologic techniques based on the release of 51Cr from labeled platelets. The amount of membrane glycoproteins (GP) IIb and IIIa in the platelets of these individuals was determined by quantitative crossed immunoelectrophoresis of Triton X-100 soluble proteins using a multispecific rabbit antibody raised against normal platelets. Platelets from the three thrombasthenic patients contained neither detectable GP IIb and GP IIIa nor detectable PlA1 antigen. Platelets from seven kindred with normal amounts of GP IIb and GP IIIa contained PlA1 antigen levels identical to those detected in platelets of normal individuals. Platelets from 13 kindred, including each parent studied, were shown to contain an amount of GP IIb and GP IIIa equivalent to 53% of that amount detected on normal platelets. Platelets from the same individuals expressed amounts of PlA1 antigen that were either 54.0 +/- 4.1 (mean +/- SD) or 28.0 +/- 2.7% of that present on platelets of normal individuals homozygous for the Al allele. The results presented in this report provide evidence that the expression of the thrombasthenic glycoprotein abnormality and the inheritance of PlA1 antigen are controlled by different genes. These results further suggest that lack of expression of the PlA1 antigen on thrombasthenic platelets results from the decrease or absence of the glycoprotein carrier of the PlA1 determinant, previously shown to be GP IIIa.     

Platelet Membrane Defects in Glanzmann''s Thrombasthenia: EVIDENCE FOR DECREASED AMOUNTS OF TWO MAJOR GLYCOPROTEINS

Platelets from patients with Glanzmann's thrombasthenia have a distinct molecular alteration of the plasma membrane surface, namely decreased amounts of a major glycoprotein designated as IIb (apparent mol wt 142,000). To identify other possible surface defects of thrombasthenic platelets, we labeled the membrane polypeptides of normal and thrombasthenic platelets by two different techniques: lactoperoxidase-catalyzed iodination and galactose oxidase oxidation, followed by reduction with tritiated sodium borohydride. Labeling patterns were determined after the polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Before the second dimension was run, platelet samples were incubated with a reducing agent, beta-mercapto-ethanol, to cleave the disulfide bonds of certain glycoproteins; the resulting changes in electrophoretic mobility permitted better resolution of individual molecules. Comparison of the labeled polypeptides of normal and thrombasthenic samples after reduction indicated decreased labeling of two major glycoproteins in thrombasthenic platelets: IIb and III (apparent mol wt 114,000). The relative proportions of radioactivity incorporated by these polypeptides were about 60 and 80% less than control values, respectively. With either Coomassie Blue or periodic acid-Schiff's reagent, glycoprotein III stained much less intensely in thrombasthenic compared to normal samples, indicating that the observed labeling deficit was caused by a decreased concentration of the molecule rather than steric inaccessibility on the membrane surface. Analysis of normal plasma membranes by affinity chromatography showed that glycoprotein IIb has receptors for lectin from Lens culinaris, the common lentil, whereas III does not. We conclude that a characteristic feature of Glanzmann's thrombasthenia is a decreased concentration of two discrete glycoproteins in the platelet plasma membrane.     

Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann''s Thrombasthenia and Bernard-Soulier Syndrome

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed ¹²⁵I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two ¹²⁵I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of ¹²⁵I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.     

A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.

To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.     

Plasminogen interacts with human platelets through two distinct mechanisms.

Glu-plasminogen, the native form of plasminogen, interacts in a specific and saturable manner with unstimulated human platelets, and the binding is enhanced fivefold by thrombin stimulation (Miles and Plow, 1985. J. Biol. Chem. 260:4303). This study characterizes the nature of the Glu-plasminogen binding sites by analyzing platelets deficient in selected proteins and functions. Platelets from patients with afibrinogenemia, Gray platelet syndrome, and the Cam Variant of thrombasthenia, a form of thrombasthenia with near normal levels of glycoprotein IIb/IIIa (GPIIb/IIIa), showed minimal augmentation of plasminogen binding to thrombin-stimulated platelets but normal binding to unstimulated platelets. This selective deficiency indicates that two distinct mechanisms are involved in the interaction of plasminogen with platelets. These abnormal platelets share a deficiency in fibrinogen. Surface expression of platelet fibrinogen, however, was not sufficient for enhanced plasminogen binding to stimulated platelets, and experiments with alpha-thrombin and gamma-thrombin indicated that fibrin formation on the platelet surface is necessary for the augmented plasminogen binding. Unstimulated and stimulated thrombasthenic platelets deficient in GPIIb/IIIa bound markedly reduced levels of plasminogen, which suggests a role for GPIIb/IIIa in plasminogen binding to unstimulated platelets. Treatment of platelets to dissociate the heterodimeric complex of GPIIb/IIIa did not significantly perturb plasminogen binding to unstimulated platelets, but the complex may be necessary for thrombin-stimulated plasminogen binding via its interaction with platelet fibrin.     

Identification of a unique type of thrombopathy of human platelets: defect in the exposure of active fibrinogen receptors in a patient with Friedreich's ataxia

Platelets of a patient with Friedreich's ataxia have been investigated because of a codiagnosis of thrombasthenia. No aggregation occurred in response to adenosine diphosphate, platelet activating factor-acether, a stimulatory antiplatelet monoclonal antibody, or phorbol myristate acetate, although platelet aggregation could be induced with thrombin, the calcium ionophore A23187, or high concentrations of collagen. Shape change, adenosine triphosphate secretion, and the responses of the platelets' protein phosphorylation systems to all agonists were normal. Immunologic analysis of the patient's radiolabeled platelet surface proteins revealed normal levels of glycoproteins IIB and IIIa. However, no iodine 125-fibrinogen binding occurred after stimulation of the patient's platelets with adenosine diphosphate. In contrast, pretreatment of the patient's platelets with the proteolytic enzyme alpha-chymotrypsin resulted in the exposure of active 125I-fibrinogen binding sites. The patient's platelets exhibited normal aggregation to fibrinogen after their pretreatment with chymotrypsin and with elastases derived either from porcine pancreas or from human granulocytes. A murine monoclonal antibody directed against the human platelet membrane glycoproteins IIb and IIIa calcium-dependent epitope and rabbit polyclonal anti-human platelet membrane and human anti-P1A1 antibodies immunoprecipitated glycoproteins IIb and IIIa and a 66 kd cleavage product of glycoprotein IIIa from sodium dodecyl sulfate-Triton X-100 extracts of the patient's proteolytically treated platelets. The patient appears to exhibit a unique type of thrombopathy involving a defect in the exposure of fibrinogen receptors. The association between the neurologic disorder and the platelet defect is still unclear.     

A variant of Glanzmann''s thrombasthenia with abnormal glycoprotein IIb-IIIa complexes in the platelet membrane.

Patient C.M. presented platelet function defects symptomatic of Glanzmann's thrombasthenia. However, analysis of surface-labeled platelets by SDS-polyacrylamide gel electrophoresis revealed the usual presence of the major glycoproteins, including GP IIb and GP IIIa. Platelet fibrinogen was not detected. Analysis of Triton X-100 extracts of Ca2+-washed C.M. platelets by crossed immunoelectrophoresis (CIE) showed normal amounts of GP IIb-IIIa complexes. However, when samples were electrophoresed through an agarose gel containing 125I-fibrinogen, the usual binding of fibrinogen to GP IIb-IIIa did not occur. Furthermore, the GP IIb-IIIa complexes showed an increased sensitivity to dissociation with EDTA, either after Triton X-100 solubilization or in the intact platelet membrane. For example, after incubation with EDTA at room temperature, the patient's platelets bound little of the monoclonal antibodies AP-2 or T10 (anti-GP IIb-IIIa complex) although normally binding Tab (anti-GP IIb). Patient C.M. appears to represent a subgroup of thrombasthenia where platelets contain unstable GP IIb-IIIa complexes unable to support fibrinogen binding.     

Thrombin binding to thrombasthenic platelets

Platelets from two patients with Glanzmann's thrombasthenia showed decreased iodination of surface glycoproteins GPIIb and GPIII. Despite these changes, the binding of [125I] alpha-thrombin to the thrombasthenic platelets was normal. Binding was linear up to a thrombin concentration of 0.1 to 0.2 U/ml, at which point a change in the slope of the binding curve was observed. At lower concentrations of thrombin, 1,000 to 2,000 molecules of thrombin were bound per platelet, with an apparent Kdiss of 0.1 to 0.3 U/ml. With high concentrations of thrombin, thrombasthenic platelets bound 30,000 to 65,000 molecules of thrombin per platelet at saturation, with an apparent Kdiss of 5 to 10 U/ml. The release of [14C]serotonin by thrombasthenic platelets as a function of thrombin concentration was also similar to release by normal platelets. These studies indicate that the receptor(s) for thrombin on the plasma membrane of platelets from patients with Glanzmann's disease are intact and that membrane glycoproteins GPIIb and GPIII play little or no role in either the initial binding of thrombin to platelets or the transmission of this surface stimulus to release-inducing mechanisms.     

Deletion of the Platelet-Specific Alloantigen PlA1 from Platelets in Glanzmann''s Thrombasthenia

Expression of a Platelet-specific alloantigen (Pl(A1)) was studied in five unrelated patients with Glanzmann's thrombasthenia using immunologic techniques based on release of (51)Cr from tagged platelets by Pl(A1)-specific antibody. Less than 1% of the normal quantity of Pl(A1) could be detected on platelets of patients 1, 2, and 3; platelets from patients 4 and 5 contained 22 and 12% of normal levels, respectively. After treatment with bromelain, platelets from patients 4 and 5, but not those from patients 1, 2, and 3, released (51)Cr as well as normal Pl(A1)-positive platelets when exposed to anti-Pl(A1). Platelets from each of the five patients reacted normally with drug-dependent antibodies and with autoantibodies specific for platelets.Polyacrylamide gel electrophoresis of thrombasthenic platelets showed marked deficiencies of glycoproteins IIbalpha and III (P < 0.0005), confirming recent reports of others. Deficiency of the two proteins as determined by gel scanning was more pronounced in patients 1, 2, and 3 than in patients 4 and 5. Normal levels of glycoproteins IIbalpha and III were found in platelets from normal subjects negative for Pl(A1).These observations are consistent with the possibility that the Pl(A1) antigen is located on one or both of the glycoproteins lacking in Glanzmann's thrombasthenia, although other explanations are possible. They further suggest that patients with thrombasthenia may be heterogeneous in respect to the degree to which these glycoproteins are deleted. The Pl(A1) antigen can be measured with considerable precision and may provide a marker useful for the diagnosis and study of Glanzmann's disease.     

Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 micrograms vWf protein/10(8) platelets. Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein Ib (GPIb) or a second against the glycoprotein IIb/IIIa complex (GPIIb/IIIa), and (b) varying the concentration of fibrinogen in the medium. The results of our studies indicate that AS-vWf binds initially to GPIb. This binding then results in the exposure of receptors for AS-vWf on GPIIb/IIIa. In the presence of plasma fibrinogen, both AS-vWf and fibrinogen bind to GPIIb/IIIa. In the presence of plasma fibrinogen, 50% more AS-vWf binds to the platelet, and this additional AS-vWf binds almost exclusively to GPIIb/IIIa. Despite this enhanced binding of AS-vWf in the absence of fibrinogen, platelet aggregation is much less than that which occurs in the presence of plasma fibrinogen. Comparative studies of AS-vWf binding to normal platelets and the platelets of patients with Glanzmann's thrombasthenia reveal decreased binding to the thrombasthenic platelets and a marked decrease in the extent of platelet aggregation. These studies indicate that AS-vWf binding to, and ensuing aggregation of, platelets is different from that observed with intact vWf protein when platelets are stimulated with either ristocetin or thrombin. The AS-vWf binds to GPIb which, in turn, makes additional AS-vWf receptors available on GPIIb/IIIa. If plasma fibrinogen is present, it competes with the AS-vWf for binding to GPIIb/IIIa and causes aggregation of platelets. In the presence of plasma fibrinogen, more of the AS-vWf binds to GPIIb/IIIa, but this AS-vWf is much less effective than fibrinogen in supporting platelet aggregation.     

Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa

Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb-GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme-treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb-GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface.     

Identification and function of the high affinity binding sites for Ca2+ on the surface of platelets.

Extracellular Ca2+ is required for platelet aggregation and secretion in response to ADP or epinephrine. Recently, we reported that the platelet surface contains two classes of high affinity binding sites for extracellular Ca2+. To identify these sites and clarify their role in platelet function, we have now (a) studied platelets congenitally deficient in surface membrane glycoproteins and (b) examined the effect of removing surface-bound Ca2+ on platelet responses to ADP and epinephrine. Unstimulated normal platelets contained 86,000 Ca2+-binding sites/platelet with a dissociation constant (Kd) of 9 nM and 389,000 sites with a Kd of 400 nM. In contrast, thrombasthenic platelets, which lack glycoproteins IIb and IIIa, exhibited a 92% reduction in the number of higher affinity Ca2+-binding sites and a 63% reduction in the number of lower affinity sites. Bernard-Soulier platelets, which lack glycoprotein Ib, were not deficient in Ca2+-binding sites. After stimulation with ADP, both normal and thrombasthenic platelets developed approximately 138,000 new Ca2+-binding sites/platelet (Kd = 400 nM), while the larger Bernard-Soulier platelets developed 216,000 new sites. These data suggest that IIb and IIIa represent the major Ca2+-binding glycoproteins on unstimulated platelets, while neither these glycoproteins nor Ib represent the new Ca2+-binding sites on stimulated platelets. Removal of Ca2+ from the platelet surface inhibited platelet function. Despite the presence of 1 mM Mg2+, ADP- and epinephrine-induced aggregation and [14C]serotonin release were markedly decreased at free Ca2+ concentrations less than 7 nM, a value similar to the Kd of the higher affinity Ca2+-binding sites. Moreover, gadolinium, a lanthanide that competed for these Ca2+-binding sites, also inhibited aggregation and serotonin release. These studies demonstrate, therefore, that the binding of extracellular Ca2+ to glycoproteins IIb/IIIa on unstimulated platelets or to additional membrane proteins on stimulated platelets is necessary for maximal platelet responses to ADP and epinephrine. Thus, the requirement for extracellular Ca2+ during platelet activation by these agonists may actually represent a requirement for surface-bound Ca2+.     

Reduced surface expression and binding of fibronectin by thrombin-stimulated thrombasthenic platelets.

Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmann's thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did induce serotonin secretion from these cells, indicating that stimulation was occurring. Thrombasthenic platelets did not inhibit thrombin-stimulated fibronectin binding to coincubated normal cells, suggesting that their defect was not due to the presence of a soluble inhibitor of fibronectin binding. Thrombin-stimulated afibrinogenemic platelets bound similar quantities of fibronectin to normal cells, indicating that the thrombasthenic deficit is not secondary to reduced fibrinogen content or binding. The thrombasthenic cells had an endogenous fibronectin content of 2.9 +/- 0.7 micrograms/10(9) platelets, whereas cells simultaneously prepared from five normal individuals contained 1.8 +/- 0.7 micrograms/10(9) platelets, a statistically insignificant difference. Nevertheless, thrombin stimulation did not increase expression of endogeneous fibronectin antigen on the surface of the thrombasthenic platelets as judged by immunofluorescence. These defects in platelet fibronectin binding and surface expression may account for some of the manifestations of Glanzmann's thrombasthenia.     

Diagnosis of Bernard-Soulier syndrome and Glanzmann''s thrombasthenia with a monoclonal assay on whole blood

Two hereditary platelet disorders, Bernard-Soulier syndrome and Glanzmann's thrombasthenia, are characterized by selective deficiencies of platelet membrane glycoproteins. Murine monoclonal antibodies were developed against platelet membrane glycoprotein Ib and against the glycoprotein IIb/IIIa complex. A rapid whole blood assay for the deficiency of these glycoproteins was developed and used to study whole blood samples from six patients with Glanzmann's thrombasthenia and three patients with Bernard-Soulier syndrome. Patients with type I and type II Glanzmann's thrombasthenia were easily detectable with this assay. This permits the diagnosis of these disorders on 200 microliters of whole blood within 2 h of blood sampling.     

Absence of platelet membrane glycoproteins IIb/IIIa from monocytes

Two-dimensional gel electrophoresis, immunoprecipitation, and crossed immunoelectrophoresis were used in the investigation of glycoproteins IIb/IIIa in platelets, monocytes, and monocyte-derived macrophages from human blood. All techniques detected the glycoproteins in platelets but not in the mononuclear phagocytes. Similar results were obtained by immunochemistry using a monoclonal antibody against the platelet glycoproteins IIb/IIIa (revealed by a gold-labeled second antibody) which bound heavily to the platelet but not to the monocyte surface. The biochemical techniques used for the analysis of mononuclear phagocytes would have reliably detected the level of glycoproteins IIb/IIIa contributed by a 5% contamination with platelets, calculated on a per cell basis. We conclude that human monocytes and monocyte-derived macrophages lack glycoproteins IIb/IIIa. Our results further indicate that centrifugal elutriation yields monocyte preparations with minimal contamination by platelets. It seems likely that the positive results obtained by other authors were due to the presence of platelets or fragments on the monocytes.     

Reconstruction of platelet proteins into phospholipid vesicles. Functional proteoliposomes.

Platelet membrane glycoproteins IIb and IIIa were reconstituted into liposomes containing phosphatidylcholine. The reconstituted vesicles bound antiplatelet antibodies and showed specific binding to thrombin-activated platelets. Prostacyclin, a known inhibitor of thrombin-activated platelet aggregation, inhibited the binding of the proteoliposomes to thrombin-activated platelets. The reconstituted vesicles also specifically bound 125I-labeled fibrinogen. This binding was insensitive to ADP but dependent on calcium ions. These data indicate that platelet glycoproteins IIb and IIIa have been successfully reconstituted into phospholipid vesicles such that their behavior is similar to that in intact platelets.     

Monoclonal antibodies to the glycoprotein IIb-IIIa epitopes involved in adhesive protein binding: effects on platelet spreading and ultrastructure on human arterial subendothelium

Platelet adherence to subendothelium depends on binding of plasma von Willebrand factor (VWF) to the subendothelial surface and its subsequent interaction with platelet membrane glycoprotein Ib (GPIb) in the Baumgartner perfusion technique. To examine the role of the platelet glycoprotein IIb-IIIa (GPIIb-IIIa) complex in these processes, we performed studies in patients with platelets deficient in GPIIb-IIIa (thrombasthenia) or GPIb (Bernard-Soulier syndrome) in the Baumgartner system with human umbilical artery segments at a wall shear rate of 2600 sec-1. Morphometry specified the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets or platelet thrombi. Total platelet adherence was defined as C + S. In thrombasthenia, C showed a small but significant increase compared with controls, whereas C + S was reduced by approximately 40%; thrombi were totally absent. With Bernard-Soulier platelets, each parameter was reduced by 72% to 93%. To verify that these findings were related to the epitopes involved in VWF, fibronectin, and fibrinogen binding, we incubated normal blood with monoclonal antibodies to the GPIIb-IIIa complex (10E5 and PLT-1) or GPIb (6D1), and these experiments yielded results similar to those observed with thrombasthenic and Bernard-Soulier platelets, respectively. By transmission electron microscopy, normal platelets preincubated with 10E5 or thrombasthenic platelets showed abnormally short and blunt pseudopodia, suggesting that the platelet GPIIb-IIIa complex plays a role in platelet spreading on subendothelium. Our observations confirm that platelet spreading is mediated at least in part by the epitope(s) of the GPIIb-IIIa complex involved in adhesive protein binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disorders of platelet function.

Platelets provide for primary hemostasis by forming a hemostatic plug at sites of vascular damage. They also provide a surface for the assembly of the coagulation protein complexes that generate thrombin, serve as a nidus for fibrin clots, and secrete factors involved in wound repair. Normal platelet function can be divided into four phases: adhesion, aggregation, secretion, and expression of procoagulant activity. Platelet adhesion initiates plug formation as platelets adhere to the connective tissue at the edges of a wound within seconds after vascular damage. When damage occurs in regions of slow blood flow, platelets adhere to subendothelial collagen, fibronectin, and laminin. However, when damage occurs in regions of rapid flow, platelet adhesion requires the presence of subendothelial von Willebrand factor (vWf) and a specific platelet receptor, the glycoprotein Ib/IX (GPIb/IX) complex. Following initial adhesion, platelets aggregate to complete the formation of a hemostatic plug. Platelet aggregation requires active platelet metabolism, platelet stimulation by agonists such as ADP, thrombin, collagen, or epinephrine; the presence of calcium or magnesium ions and specific plasma proteins such as fibrinogen or vWf; and a platelet receptor, the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. Thus, platelet stimulation results in the generation of intracellular second messengers that transmit the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or vWf) then binds to GPIIb/IIIa and crosslinks adjacent platelets to produce platelet aggregates. Platelet stimulation also results in platelet secretion and the elaboration of platelet procoagulant activity. During secretion, substances are released to propagate the aggregation response and to promote wound healing; the expression of procoagulant activity localizes thrombin generation to the site of vascular damage. Disorders of platelet function can be divided into those of congenital and those of acquired origin. Although congenital disorders are uncommon, acquired disorders are encountered frequently in clinical practice. Congenital absence of GPIb/IX and GPIIb/IIIa results in the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT), respectively. Each is an autosomal recessive bleeding disorder in which absence of a protein complex renders the affected platelets incapable of undergoing either vWf-mediated adhesion (BSS) or fibrinogen-mediated aggregation (GT).(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombin-induced platelet membrane glycoprotein IIb and IIIa complex formation. An electron microscope study

The topographic relationships of platelet membrane glycoprotein IIb and glycoprotein IIIa have been studied in stimulated and unstimulated human platelets using immunoelectron microscopy. An indirect approach with ferritin-conjugated goat anti-rabbit gamma-globulin was used to localize the rabbit antibody to glycoprotein IIIa. The second ultrastructural label was keyhole limpet hemocyanin conjugated directly to antibody to glycoprotein IIb. Using the double labels, it was demonstrated that glycoprotein IIb and glycoprotein IIIa were distributed randomly in the unstimulated platelet membrane. After platelet stimulation with thrombin, large clusters of glycoprotein IIb- glycoprotein IIIa complexes were formed. No complex formation between glycoprotein Ib and glycoprotein IIb was observed in control experiments. These observations suggest that thrombin stimulation initiates the specific glycoprotein IIb-glycoprotein IIIa macromolecular complex formation on the platelet surface, which may act as the active fibrinogen-binding site required for normal platelet aggregation.