Kaese, S E

Abstract We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

Diagnosis of Bernard-Soulier syndrome (BSS) is complicated by the difficulty of separating the giant platelets from other blood cells to pursue analyses of platelet function and structure. We report on the utility of three whole blood assay techniques for diagnosis of a patient with BSS. To our knowledge, these three techniques have not been simultaneously applied or compared for efficacy in laboratory diagnosis of BSS. (1) Whole blood platelet aggregation responses, studied with an electrical impedence aggregometer, were equivalent to those more laboriously obtained using platelet-rich plasma prepared by unit gravity sedimentation, studied with an optical light transmittance aggregometer. Platelet aggregation responses were normal with ADP or collagen stimulation, and absent with Ristocetin or bovine plasma stimulation. (2) Whole blood radioimmunoassay of platelet glycoprotein (GP) expression was performed using iodinated murine monoclonal antibodies HP1-1D (anti-GP IIb/IIIa) and 6D1 (anti-GPlb, kindly supplied by Dr. Barry Coller, Stony Brook, NY). After incubation with citrated whole blood, centrifugation was used to separate cell-bound antibody which was quantitated with a gamma counter. The patient’s whole blood had a normal level of cell-bound GP Ilb/IIIa, but a markedly reduced level of cell-bound GP lb (5% of normal mean; n = 20). (3) Whole blood smear immunocytochemical staining with the monoclonals (indirect immuno-alkaline phosphatase technique), and qualitative analysis by light microscopy, revealed a marked reduction of GP lb expression by the patient’s giant platelets, whereas GP Ilb/IIIa expression was normal. This latter technique might be especially valuable as a screening technique when the patient is not directly available for laboratory study. Together with the patient’s life-long history of thrombocytopenia and moderate bleeding diathesis, and other laboratory observations including markedly prolonged bleeding times and reduced whole blood prothrombin consumption, these data established diagnosis of BSS. We conclude that these three relatively simple assays of platelets in whole blood should be of particular value in the laboratory differential diagnosis of patients with congenital thrombocytopenias and giant platelet syndromes.

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.

A 39-year-old 75 kg man with Type I Glanzmann's thrombasthenia developed recurring severe therapy-refractory nosebleeds, associated with nasal septal deformity, synechial scarring, and previous radiotherapy. During a 21 month period he required 16 hospitalizations for treatment of severe epistaxis. At initial hospitalization it was observed that his epistaxis ceased shortly after transfusion of 10 bags (units) of plasma cryoprecipitate (cryo), although transfusion with HLA-matched apheresis platelets from 3 donors, and topical therapy (cautery, packing), had been ineffective. Serum anti-platelet antibodies were not detectable by indirect immunofluorescence. During 9 subsequent hospitalizations his epistaxis stopped promptly (usually within 1 hour) following cryo transfusion (10 or 20 bags), including 6 occasions when cryo was the only therapy. On 6 additional occasions his epistaxis did not stop following cryo transfusion, but did stop after subsequent platelet transfusions or topical therapy. Eventually he underwent nasal septal reconstructive surgery surgery, and severe epistaxis has not recurred. Hemostatic studies, before and after cryo transfusions on 5 occasions, did not show improvement of platelet aggregation defects nor of Ivy bleeding times, although occasionally the volume of blood emanating from bleeding time punctures appeared decreased following cryo transfusion. Platelet glycoprotein (GP) IIb/IIIa antigen was measured in aliquots of 10 of the pools of cryo received by the patient (representing 100 bags of total volume 2200 ml), using an immunoradiometric assay (Nichols et al, Blood 68:300a, 1986). On average, the transfused cryo pools contained GP IIb/IIIa equivalent to 1.1 x 108 platelets/ml (range 0.6-1.7 x 108/ml. Our recently reported studies of blood bank cryo documented similar GP IIb/IIIa levels, and revealed that >93% of GP IIb/IIIa in cryo is present in the form of sedimentable membranous platelet macroparticles and microparticles. We hypothesize that the GPIIb/IIla-bearing platelet particles in transfused cryo might account for the improvement in hemostasis we frequently observed. We conclude that cryo transfusion deserves further study as a potentially useful therapeutic adjunct in promoting hemostasis in individuals with Glanzmann's thrombasthenia.