In vitro characterization of a monoclonal IgG(kappa) from a patient with planar xanthomatosis.

OBJECTIVE: To characterize a monoclonal IgG(kappa) (MAb) from a patient with planar xanthoma that precipitated with serum lipids at reduced temperature. METHODS: The molecular weight (Mr), sensitivity to proteases, and glycosylation of the purified MAb were analyzed. The specificity of the MAb was tested by measuring cryoprecipitation with pure high- (HDL) and low-density (LDL) lipoproteins. The effect of choline, phosphocholine, and glycerol 3-phosphate on the precipitation temperature of LDL by the MAb was studied. RESULTS: The MAb was larger than normal IgG due to hyperglycosylation of the MAb light chain. It formed cryoprecipitates with pure HDL and LDL as well as the lipids extracted from these lipoproteins. Fab fragments of the MAb lowered the temperature of its precipitation with LDL. Choline, phosphocholine, and glycerol 3-phosphate also lower the precipitation temperature. CONCLUSION: This is the first human IgG reported with apparent specificity for phosphocholine antigens. Its precipitation with lipids at reduced temperature suggests that it recognizes conformations of phospholipid head groups that develop below core body temperature.

Plasminogen interaction with platelets: the importance of carboxyterminal lysines.

INTRODUCTION: Thrombin stimulation enhances plasminogen binding to platelets and promotes platelet-dependent plasmin generation. The objective of this study was to determine whether carboxyterminal lysines (C-lysines) are important for these processes, as they are in other cell types. MATERIALS AND METHODS: 125I-plasminogen and varying concentrations of unlabeled plasminogen were added to washed platelets that were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. In some experiments the platelets were digested with carboxypeptidase B to remove C-lysines. Platelet-dependent plasmin generation was also studied by adding plasminogen and tissue plasminogen activator to platelet suspensions and monitoring the conversion of a plasmin specific chromogenic substrate. The cells were either resting or stimulated with thrombin, thrombin receptor activating peptide, or ADP. The effect of the thrombin inhibitor lepirudin and the plasmin inhibitor aprotinin on plasminogen binding and the appearance of C-lysines was also investigated. RESULTS: Thrombin, but not thrombin receptor activating peptide or ADP, stimulated high-affinity binding of plasminogen and greatly promoted platelet-dependent plasmin generation. Digestion with carboxypeptidase B eliminated thrombin-induced high-affinity binding and reduced thrombin-induced plasmin generation by increasing the Michaelis constant. Lepirudin, but not aprotinin, inhibited thrombin-stimulated plasminogen binding to platelets. CONCLUSION: C-terminal lysines are necessary for high-affinity binding of plasminogen to platelets and for platelet-supported plasmin generation. The origin of the C-lysines is not clear, but they may result from a direct effect of thrombin, rather than an intermediate enzyme such as plasmin.

Is thyroid hormone suppression therapy prothrombotic?

The purpose of this study was to determine whether chronic thyroid hormone suppression therapy (THST) is prothrombotic. We obtained blood samples from 14 thyroid cancer patients while on THST and after they had become hypothyroid for radioiodine whole-body scanning and therapy. Prothrombin fragment 1 + 2, fibrinogen, factor VIII, antithrombin, tissue plasminogen activator antigen (tPA), plasminogen activator inhibitor 1 (PAI-1), PAI-1/tPA, and C-reactive protein were significantly (P < 0.05) higher in the hyper- than in the hypothyroid state, whereas protein C and plasmin-antiplasmin complexes were significantly lower during the hyperthyroid period. When the 10 female patients were hyperthyroid, their levels of prothrombin fragment 1 + 2, fibrinogen, protein S, antithrombin, tPA, PAI-1, and PAI-1/tPA were significantly higher (P

Parameters of coagulant and fibrinolytic capacity and activity in postmenopausal women: within-subject variability.

We have analyzed the within-subject variability of a battery of parameters of coagulant and fibrinolytic capacity and activity in postmenopausal women. We observed large differences in within-subject variability among the tests and have demonstrated how such data can be used to estimate the number of times a parameter must be measured to produce a statistically adequate sample.

PF4 ENHANCED assay for the diagnosis of heparin-induced thrombocytopenia in complex medical and surgical patients.

OBJECTIVE: To evaluate the sensitivity and specificity of the PF4 ENHANCED (GTI Diagnostics, Waukesha, WI) enzyme-linked immunosorbent assay for heparin-induced thrombocytopenia using the carbon-14 serotonin-release assay as the reference method. DESIGN: A total of 34 patients were prospectively enrolled with a variety of diagnoses and suspected heparin-induced thrombocytopenia. They were clinically scored and underwent testing with the (14)C-serotonin-release assay. Enzyme-linked immunosorbent assay and (14)C-serotonin-release assay results were also available from 21 medical and surgical patients who had previously been tested. MAIN RESULTS: With the (14)C-serotonin-release assay as the reference method, the sensitivity and specificity of the enzyme-linked immunosorbent assay were 93% and 65%, respectively. There was only one false-negative enzyme-linked immunosorbent assay. The clinical scores were frequently misleading, largely because of difficulty excluding other causes of thrombocytopenia. CONCLUSION: Because of its high sensitivity, we believe the PF4 ENHANCED enzyme-linked immunosorbent assay should be used to identify heparin-induced thrombocytopenia in patients with multiple potential causes of thrombocytopenia, although false-positive results will not be uncommon.

The impact of major surgery on blood coagulation factors and thrombin generation.

We studied the blood coagulation system of 14 patients with metastatic malignancies before and after they had undergone major surgery. In addition to measuring a battery of coagulation factors, we assessed the function of the system with assays of whole blood thrombin generation. With the exceptions of factor VIII (fVIII), which increased, and fibrinogen and fIX, which did not change, the activities of all the pro- and anticoagulant proteins were significantly lower postoperatively. However, the thrombin generating capacity of the system was relatively preserved. Although the integral of thrombin activity over time was lower after surgery, the mean peak thrombin concentration was unchanged and the time to clot formation was shortened. Similar changes could be reproduced by lowering the concentrations of pro- and anticoagulant factors together in control blood samples. Therefore, simultaneous reductions in pro- and anticoagulant proteins postoperatively worked to maintain the functional integrity of the blood coagulation system.

The effect of red blood cells on thrombin generation.

We have developed an assay of thrombin generation in clotting whole blood. Because our goal was to study patients with red blood cell diseases that are associated with thrombosis, the initial evaluation of this assay analysed the effect of the haematocrit and haemolysate on the total amount of thrombin activity generated and the maximal concentration of thrombin achieved. Both of these parameters were proportional to the haematocrit throughout a wide range of clinically relevant cell concentrations. Red cell lysate also augmented thrombin generation. Because this effect was removed by filtration of the lysate, we propose that it was related to red cell microparticles.