A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100-solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde-fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard-Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.     

Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with Bernard-Soulier syndrome.

Three distinct gene products, the alpha and beta chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. We have analyzed the molecular basis of this disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ib alpha but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ib alpha, which by lacking half of the sequence on the carboxyl-terminal side, including the trans-membrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ib alpha allele. Nonsense mutation and truncated GP Ib alpha polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ib alpha alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome.     

Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets

The interaction of the heparin-dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin-induced thrombocytopenia. Both normal platelets and Bernard-Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib. GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV.3) strongly inhibited the heparin-dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti-GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti-GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C-serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin-dependent antibody with platelets and heparin is mediated by a Fc-dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti-GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.     

Heparin-induced thrombocytopenia: further studies of the effects of heparin-dependent antibodies on platelets

Studies were performed with the sera of five patients with heparin-induced thrombocytopenia. Each patient serum was found to cause heparin-dependent aggregation of normal and Bernard-Soulier syndrome platelets but not Glanzmann's thrombasthenia or fixed normal platelets. This antibody effect was blocked by inhibition of both aerobic and anaerobic metabolism and an increase in intraplatelet c-AMP with prostaglandin E1. These findings suggest that the antibody-induced platelet aggregation was not a passive agglutination of cells but an active process requiring metabolic energy and membrane glycoproteins IIb/IIIa but not glycoprotein Ib. In addition, platelet aggregation by four patient sera was inhibited by aspirin or apyrase indicating a prostaglandin synthesis and ADP-dependent mechanism and the reaction could be suppressed by dipyridamole alone. However, platelet aggregation induced by the serum of one patient was only inhibited partially by the combination of aspirin and apyrase suggesting mediation of the antibody effect in part by a pathway independent of extracellular ADP and prostaglandin synthesis and this antibody reaction could only be completely suppressed by a combination of aspirin and dipyridamole. Heterogeneity exhibited by these antibodies may influence the choice and effectiveness of antiplatelet therapy in heparin-induced thrombocytopenia.     

A case of Bernard-Soulier syndrome: study of platelet glycoprotein Ib in a kindred

The Bernard-Soulier syndrome is characterized by low platelet counts, abnormally large (giant) platelets, and impaired or absent platelet aggregation by the inducer antibiotic ristocetin. The recent discovery of the inherited biochemical defect and the deficient synthesis of platelet glycoprotein Ib (GP-Ib), has contributed greatly to the understanding of the disease. We report a case of the Bernard-Soulier syndrome presenting with bleeding from the pharynx after adenotomy. The patient and nearest family members were studied by a novel immunoperoxidase method for quantification of platelet glycoprotein Ib using a specific monoclonal antibody (AN51).     

Thrombocytopenia associated with pregnancy in a patient with type IIB von Willebrand's disease

Thrombocytopenia may accompany variant (type IIB) von Willebrand's disease (vWD) and is thought to result from binding of the abnormal von Willebrand factor (vWF) to the patient's platelets with subsequent platelet aggregate formation and clearance. We have studied a patient with type IIB vWD who became thrombocytopenic during two pregnancies. During the third trimester of pregnancy, her platelet counts dropped to 20,000 to 30,000/microL, and an increase in the intermediate-sized vWF multimers was seen on agarose gel electrophoresis. During this time her platelet-rich plasma showed spontaneous platelet aggregation, and her plasma caused spontaneous aggregation of normal washed platelets. Antibody to platelet glycoprotein Ib completely blocked the spontaneous platelet aggregation, while antibody to platelet glycoprotein IIb/IIIa did not block the response at the concentrations used. Inhibitors of platelet function that elevate platelet cyclic AMP also blocked the response, but aspirin had no effect on the spontaneous platelet aggregation. The patient illustrates that the platelet counts in one individual can vary greatly in type IIB vWD and that the thrombocytopenia that occurs can appear under physiologic conditions that stimulate the endogenous production of the patient's abnormal vWF. The mechanisms leading to spontaneous platelet aggregation and thrombocytopenia appear to be similar to those described for other patients with type IIB vWD.     

Platelet aggregating IgG antibody to platelet surface glycoproteins associated with thrombosis and thrombocytopenia

Previously described platelet-aggregating antibodies associated with thrombosis and thrombocytopenia required heparin for their in vivo and in vitro expression. We have observed a patient with thrombosis who became thrombocytopenic during heparin treatment, but who suffered further thrombotic events and continued thrombocytopenia for 3 months after heparin withdrawal. The patient's plasma contained a potent platelet aggregating factor reactive with both his own and normal platelets in the absence of heparin. It also caused [14C]serotonin secretion from labelled platelets from normal donors and patients with either Glanzmann's thrombasthenia or Bernard-Soulier syndrome. This factor was an IgG and was neutralized by antibody specific for IgG lambda light chains. While the patient was thrombocytopenic an IgG paraprotein with lambda light chains was detected by isoelectrofocussing. After corticosteroid treatment it disappeared and the patient recovered. The active, but not the recovery serum contained IgG which immunoprecipitated a glycoprotein with characteristics of Glycoprotein IV from platelets labelled with Na[3H]BH4/periodate. Thus platelet-aggregating IgG antibodies with direct specificity for platelet surface glycoproteins may be associated with thrombosis/thrombocytopenia.     

Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.     

Enhanced botrocetin-induced type IIB von Willebrand factor binding to platelet glycoprotein Ib initiates hyperagglutination of normal platelets

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPIb induced by these two substances have been reported. We have recently shown that the GPIb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.     

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.     

Bernard-Soulier syndrome: diagnosis by an ELISA method using monoclonal antibodies in 2 new unrelated patients

Two unrelated patients with Bernard-Soulier syndrome and their relatives were studied. The patients demonstrated severe bleeding diathesis, the relatives were asymptomatic. The propositi showed the characteristic abnormalities of the syndrome: thrombocytopenia, a percentage of giant platelets higher than 65%, prolonged bleeding time and defective platelet aggregation to ristocetin and bovine plasma. On the contrary, in the heterozygotes, the typical abnormalities were not fully evident. We introduce a simple ELISA method for the precise definition of both homozygous and heterozygous states for the syndrome by the quantitation of platelet glycoprotein (GP) Ib. Specific binding of monoclonal antibodies anti-platelet GPIb was performed both by direct binding of radioiodinated antibody and by ELISA. Comparable results were obtained. In fact, we demonstrated near absence of GPIb in the 2 propositi and about half the amount in the heterozygotes studied compared to normal platelets.     

Acquired Bernard-Soulier syndrome: a case with necrotizing vasculitis and thrombosis

We describe a patient with positive antinuclear antibodies, polyclonal gammopathy and high level of circulating immunocomplexes, resulting in vascular purpura. In addition, the patient had a slightly prolonged bleeding time and an isolated defect of ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). The patient's plasma also inhibited RIPA in normal PRP and in normal platelet suspension. The activity and multimeric structure of plasmatic von Willebrand factor showed no alteration. We could demonstrate an autoantibody against platelet membrane glycoprotein (GP) Ib, using an ELISA-type assay. These data suggest an acquired Bernard-Soulier syndrome. We suggest that the patient had an immunocomplex-mediated leukocytoclastic vasculitis accompanied by production of antinuclear autoantibodies as well as the presence of an autoantibody against GPIb. The titer of the anti-GPIb antibody, however, was too low to induce significant platelet-type bleeding tendency, only laboratory alterations were found. Moreover, in a later stage of her disease, she developed a severe necrotizing vasculitis which was followed by a deep venous thrombosis.     

An inherited macrothrombocytopenic disorder with abnormal large granules

Inherited giant platelet syndromes are a heterogeneous group of rare bleeding disorders. In the current study, a patient was reported with prolonged bleeding time, thrombocytopenia and giant platelets. Both the patient's red and white cell counts were normal without morphological abnormalities. The electron microscopy of platelets showed abnormal large electron-dense granules in the cytoplasm with exocytosis being easily observed. Her fathers platelets had the same abnormalities. The expression of glycoprotein (GP)Ib, GPIIb and GPIIIa on the surface of the patient's platelets was normal, and her platelet aggregation in response to ADP and ristocetin was also normal. Immunogold probes combined to monoclonal antibodies against GPIIIa, P-selectin or CD63 could not identify any relationship between the aberrant structures and alpha granules or lysosomes of platelet. Serotonin level was normal in the platelets of the patient, indicating the electron opaque granules are not dense bodies. The morphological abnormalities of the platelets from the patient are clearly distinguishable from other hereditary giant platelet disorders. We propose that the abnormal large granules from the patient's platelets probably represent a novel inherited thrombocytopenic disorder.     

Platelet aggregation induced by type IIb platelet von Willebrand factor

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

Bernard-Soulier Disease: a Study of Four Patients and their Parents

S ummary. Two families with Bernard-Soulier disease, including four patients and three of their parents, were studied and detailed clinical summaries are presented. One patient in each family has suffered severe bleeding problems while the other affected sibling is less severely affected. There has been no excessive bleeding in any of the parents or other family members. The patients demonstrated the abnormalities characteristic for Bernard-Soulier disease: thrombocytopenia, giant platelets, prolonged bleeding time, abnormal platelet aggregation to human FVIII_vWF and ristocetin or bovine FVII_vWF alone, defective ristocetin-induced binding of human ¹²⁵I–FVIII_vWF multimers, decreased platelet lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. The parents had normal platelet counts, bleeding times, and FVIII-mediated aggregation. However, the parents had anormally large platelets, decreased sensitivity to lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. Therefore the heterozygous state for Bernard-Soulier disease is recognizable by platelet membrane abnormalities although there is no defect of platelet function and no excessive bleeding. Red cell membrane proteins of one patient were normal, suggesting that phenotypic expression of the Bernard-Soulier disease defect is restricted to platelets.     

Plasma of patients with Upshaw-Schulman syndrome, a congenital deficiency of von Willebrand factor-cleaving protease activity, enhances the aggregation of normal platelets under high shear stress

Upshaw-Schulman syndrome (USS) is an autosomal recessive disorder characterized by repeated episodes of chronic thrombocytopenia and microangiopathic haemolytic anaemia (MAHA) that responds dramatically to infusions of fresh frozen plasma (FFP). Recent studies have provided consistent evidence that USS is a congenital deficiency of plasma von Willebrand factor-cleaving protease (VWF-CPase) activity and, therefore, unusually large VWF multimers (UL-VWFMs) are present in the plasma. However, the molecular mechanism of the clinical symptoms of USS is not well understood. We studied the relationship between UL-VWFMs and thrombocytopenia in two USS patients by analysing platelet aggregation using a mixture of the patient's plasma and normal washed platelets under high shear stress. Our results clearly showed a remarkably enhanced high shear stress-induced platelet aggregation (H-SIPA) by the patient's plasma. At 24 h after FFP infusion (approximately equal to 10 ml/kg body weight), the enhanced H-SIPA became almost completely normalized but, 2 d later, it began to return to the preinfusion level. These results were in accordance with the change in VWFM patterns. The specific effects of enhanced H-SIPA on VWF, platelet glycoprotein Ib and endogenous ADP released from platelets upon stimulation were confirmed using reagents that specifically inhibit their respective functions. Our present results clearly indicate that thrombocytopenia in USS patients is caused by a combination of the presence of UL-VWFMs, platelets and high shear stress generated in the microcirculation.     

The mechanism of heparin-induced platelet aggregation

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

Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis

The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS- polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two- dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.     

Heparin-induced thrombocytopenia: laboratory studies

This report describes studies into the pathophysiology of heparin- induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin- induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule.