alpha-chymotrypsin and Blood-Platelet-Disorders

The platelet-binding characteristics of three different antibodies that completely block adenosine diphosphate (ADP)-induced platelet fibrinogen binding and react with glycoproteins IIb, IIIa, or both, were studied. Two of the antibodies are murine monoclonal antibodies (10E5 and 7E3), and the third is an alloimmune antibody produced by a patient with Glanzmann's thrombasthenia who has received multiple transfusions (E.S.). The two monoclonals differ in that 7E3, but not 10E5, binds to dog platelets as well as to human platelets. In mutual competition studies, 7E3 did not interfere with 10E5 binding, indicating that both antibodies could bind to the complex simultaneously. E.S. IgG produced only minor inhibition of 10E5 binding, but nearly complete inhibition of 7E3 binding, suggesting that its epitope lies closer to the 7E3 epitope than the 10E5 epitope. Ethylenediaminetetraacetic acid (EDTA) pretreatment of intact platelets at 22 degrees C and pH 7.75 did not affect 10E5 binding, but significantly inhibited 7E3 binding. Similar treatment of intact or solubilized platelets at high pH and temperature produced splitting of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex and loss of both 10E5 and 7E3 binding. The 10E5 bound equally well to unactivated and activated platelets, and even rapid fixation of whole blood produced only a modest decrease in 10E5 binding. Preincubation of platelets with either E.S. IgG or 10E5 partially prevented EDTA from splitting the complex, and both monoclonal antibodies were able to bind to such complexes despite the EDTA treatment. These studies indicate that the binding of antibodies to several different epitopes on the GPIIb-IIIa complex can result in similar functional properties in blocking fibrinogen binding and platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate; Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Blood Platelet Disorders; Blood Platelets; Chymotrypsin; Dinoprostone; Dogs; Epitopes; Epoprostenol; Fibrinogen; Glycoproteins; Humans; Immunoglobulin Allotypes; Iraq; Israel; Membrane Proteins; Platelet Aggregation; Platelet Membrane Glycoproteins; Prostaglandins E; Radioimmunoassay; Receptors, Cell Surface; Thrombasthenia

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.

Topics: Antibodies, Monoclonal; Binding Sites, Antibody; Blood Platelet Disorders; Blood Platelets; Cell Membrane; Chymotrypsin; Fibrinogen; Glycoproteins; Humans; Membrane Proteins; Platelet Aggregation; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Thrombasthenia

Topics: Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Cell Membrane; Chymotrypsin; Fibrinogen; Humans; Kinetics; Platelet Aggregation; Platelet Membrane Glycoproteins; Receptors, Cell Surface

Although 125I-fibrinogen becomes associated with washed platelets from normal human subjects during ADP-induced shape change and aggregation, 125I-fibrinogen did not become associated with washed plateletes from a thrombasthenic subject during ADP-induced shape change and the platelets did not aggregate. Platelets from control and thrombasthenic subjects were treated with chymotrypsin, which is known to degrade platelet membrane glycoproteins. More 125I-fibrinogen became associated with chymotrypsin-pretreated platelets from normal subejcts than with untreated platelets, and fibrinogen caused the enzyme-treated platelets to aggregate. 125I-fibrinogen did not become associated with chymotrypsin-pretreated thromobasthenic platelets, and fibrinogen did not aggregate them. Thus, there appears to be a defect in thrombasthenic platelets that prevents the association of fibrinogen with them.

Topics: Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Chymotrypsin; Collagen; Fibrinogen; Humans; Iodine Radioisotopes; Platelet Aggregation; Serotonin; Thrombin

The platelet membrane receptor for quinidine- and quinine-dependent antibodies was studied in three patients with the Bernard-Soulier syndrome (BSS) and in normal subjects with immunologic techniques based on the release of 51Cr from labeled platelets. The receptor could not be detected on BSS platelets but was present on platelets from each of 180 normal subjects. BSS platelets reacted normally with other allo- and autoantibodies. In confirmation of previous reports, BSS platelets were found to be deficient in glycoproteins Ib and Is. However, after apparently total cleavage of these proteins from the membrane of normal platelets by controlled hydrolysis with trypsin or chymotrypsin, 80% of the drug-dependent antibody receptor activity was retained. These observations suggest the existence of an additional, hitherto unrecognized membrane defect in Bernard-Soulier platelets.

Topics: Antibodies; Binding Sites; Blood Platelet Disorders; Blood Platelets; Blood Proteins; Cell Membrane; Chymotrypsin; Glycoproteins; Humans; Membrane Proteins; Quinidine; Quinine; Syndrome; Trypsin

Topics: Blood Platelet Disorders; Blood Platelets; Blood Proteins; Chymotrypsin; Fibrinogen; Humans; Latex; Microspheres; Platelet Aggregation; Serotonin; Serum Albumin, Bovine; Time Factors