Michael W. Stewart

Heparin-induced thrombocytopenia: an improved method of detection based on lumi-aggregometry

Summary Heparin‐induced thrombocytopenia (HIT) is a recognized complication of heparin administration. Early detection of this syndrome is essential in the prevention of immune‐mediated thromboembolic sequelae. The 14C‐sero‐tonin release assay (SRA) has been used in reference laboratories to identify sera from patients on heparin therapy capable of inducing platelet dense granule release. In an attempt to improve existing methodologies, we employed luminographic detection of platelet‐dense granule ATP release as an endpoint of HIT antibody‐mediated platelet activation. Sera tested included 10 SRA confirmed positive and five SRA confirmed negative samples (to establish the assay), five samples from patients with thrombocytopenia not on heparin therapy and 34 patients suspected of HIT syndrome. All SRA confirmed positive sera (n= 19) were positive by the luminographic procedure. 24/26 SRA confirmed negative sera and five sera from thrombocytopenic patients not on heparin therapy were negative using luminography. Two of four sera yielding equivocal SRA results were found to be positive by the luminographic technique. The data suggest that the use of a lumi‐aggregometer in the coagulation laboratory to detect HIT antibody‐induced platelet activation is a reliable alternative to the SRA. The luminographic procedure is both rapid and sensitive, and does not require the use of biohazardous radio‐isotopes.

GLUT-3 (brain-type) glucose transporter polypeptides in human blood platelets

Antiserum directed against a C-terminal peptide of the human GLUT-3 (brain type) equilibrative glucose transporter isoform reacted with polypeptides M(r) 46,000-48,000 on immunoblots of human platelets. Photoirradiation of human platelets in the presence of 3H-cytochalasin B led to radiolabeling of polypeptides of identical mobility. This labeling was substantially reduced by preincubation the cells with 440 mM D-glucose, but not 440 mM L-glucose, consistent with glucose transporter function. Only traces of GLUT-1 (erythrocyte type) glucose transporter polypeptides were detected, and there was no evidence of GLUT-2 (liver type) transporter on immunoblots of platelet proteins.

Antiphospholipid antibody-dependent C5b-9 formation

The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b‐9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat‐inactivation of the sera. The response was restored if the heat‐inactivated APA‐positive sera were supplemented with normal sera. Adsorption of the APA‐positive sera with phospholipid (PL)‐coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b‐9 (aE11) also inhibited platelet destruction, suggesting that the APA‐dependent platelet destruction might be complement‐mediated. Purified APA, in the presence of normal serum, induced C5b‐9 formation and binding to PL‐coated beads in a dose‐dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b‐9 production showed that all sera testing negative for the presence of APA also tested negative for C5b‐9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b‐9 production. Sera with low or moderate GPL values showed varying levels of C5b‐9 production. These data suggest that complement may play a key role in APA‐dependent platelet activation, in vivo.

Rapid detection of anticardiolipin antibodies

A rapid screening method for the detection of antiphospholipid antibodies is described. Dense, red dyed polystyrene beads coated with cardiolipin were incubated with test sera for a short period of time, then added to a microtube containing anti‐human IgG in a gel provided within a pre‐cast card (DiaMed ID Microtyping System). The card was centrifuged at 150g for 5 min and then examined for movement of the beads through the gel. Beads without bound antibody travelled through the gel and formed a pellet on the bottom of the tube. Anti‐human IgG within the gel matrix impeded cardiolipin‐coated beads when antiphospholipid antibodies bound to the beads. Positivity was indicated by the formation of a layer of beads on the top of the gel matrix. Prospective analysis of 103 samples for the presence of antiphospholipid antibodies by flow cytometry and the gel‐card technique showed good correlation between the two methods. All samples found to be positive by flow cytometry (23 of 103) were identified as positive by the gel‐card technique. Two samples were identified as positive by the gel‐card method but negative by flow cytometry. The technique is simple to perform and should prove useful as a rapid screening method for the detection of antiphospholipid antibodies. Am. J. Hematol. 57:315–319, 1998.

Platelet activation by a novel solid‐phase agonist: effects of VWF immobilized on polystyrene beads

The interaction between platelets stirred in suspension and VWF immobilized on polystyrene beads was studied. Platelets aggregated and released ATP in response to stirring with VWF beads. Closer examination of the interaction using transmission electron microscopy revealed that the platelets did not simply aggregate with one another but initially adhered to the beads and spread. Platelets in suspension then bound to the bead‐adherent platelets forming layers of platelets associated with each bead. The VWF bead‐induced platelet activation was com‐pletely inhibited by addition of monoclonal antibody (mAb) to GPIb or GPIIb/IIIa. In addition, the activation response was inhibited in the presence of aspirin, indomethacin or the thromboxane receptor antagonist BM13.177, demonstrat‐ing a dependence on an intact cyclo‐oxygenase pathway.  Platelet function studies were carried out on 30 patients with a history of mild bleeding using conventional optical aggregation and VWF bead‐induced platelet activation. 12 patients were abnormal by conventional optical aggregometry, whereas 27 patients showed depressed ATP release in response to VWF beads. The results suggest that easily‐bruised patients may have a platelet function defect rather than a vascular‐based abnormality and that VWF bead‐induced platelet activation is a more sensitive test for detecting platelet dysfunction.

Assessment of ω-fatty-acid-supplemented human platelets for potential improvement in long-term storage

Abstract Uptake of omega (ω)−3 fatty acids can influence membrane stability and cell mobility. We investigated the effects of ω−3 and −6 fatty acids on the hemostatic efficacy of human platelets using an in vivo rabbit bleeding model. In vitro assays such as platelet aggregation, vWF bead-mediated ATP release and platelet adhesion to beads (measured by the residual platelet count [RPC] {free platelet count after reacting with the beads}/{baseline platelet count}×100=%RPC; a high %RPC indicates reduced platelet function) were conducted on platelets treated with 1% fish oil (ω−3); 2% fish oil emulsion or 1% soy oil (ω−6). Oil treatment of platelets reduced the vWF bead-induced ATP release insignificantly. Addition of ω−3 agents reduced physical reactivity (%RPC) with the vWF beads by a factor of 1.2 (oil) and 1.9 (emulsion). The ω−6 oil enhanced reactivity by a factor of 1.7. After washing to remove excess reagent, platelet resuspension was most efficient with the ω−3 emulsion. Platelet function was higher with the ω−3-treated platelets (%RPC=52.3%, ω−3 oil; 63.3%, ω−3 emulsion vs. 85%, ω−6 oil; 82% untreated platelets). Ethyl-palmitate-treated thrombocytopenic rabbits were infused with human platelets. Survival times of the treated platelets, as monitored by flow cytometry (6.2–8.2 h) were comparable to untreated platelets (8.6 h). In the rabbit kidney injury model, blood loss after infusion of the treated platelets was similar to that of saline-infused rabbits (75.3±3.4 g). However, platelets washed prior to infusion reduced blood loss to a value comparable to that of fresh platelets (48.3±5 g). Furthermore, the presence of the infused platelets at the injury site was clearly visualized using FITC-tagged anti CD42a antibody. Thus, the ω−3-based agents protect the platelets from damage during the washing procedure as demonstrated in vitro by improved platelet resuspension, low %RPC, high stimulus-responsive ATP secretion and a reduction in blood loss in vivo.

Evaluation of hemostatic activity of desamino-D-arginine vasopressin (DDAVP) in uremic rats

DDAVP, an analog of vasopressin, has been shown to have hemostatic activity. Although it has been used clinically to control bleeding, there have been very few attempts to characterize this agent in experimental animals. We describe a new animal model which is fast, reliable and inexpensive, and which may be used to screen novel analogs of the peptide. Under sodium pentobarbital anesthesia, rats were bilaterally nephrectomized and implanted with indwelling intravenous cannulae. The next day they were re-anesthetized, a standardized cut was made in the tail and the tail was allowed to bleed into warm isotonic saline (25 ml). After 10 min., the tail was removed from the saline and the blood loss was measured either by laser nephelometry or by colorimetric analysis. DDAVP was then injected intravenously and the rat was allowed to rest quietly for 30 min., after which time a second incision was made in the tail and blood loss again measured for 10 min. Unlike bleeding time which was highly variable, blood loss proved to be a reliable index of the hemostatic activity. Thus we were able to demonstrate that DDAVP reduced blood loss in the uremic rats, whereas it was without effect in intact rats.

A competitive ELISA technique for the measurement of von Willebrand factor antigen (vWF:Ag) using staphylococcal protein A peroxidase.

A competitive ELISA technique for measurement of von Willebrand factor antigen (vWF:Ag) using Staphylococcal Protein A peroxidase is described. The standard used in this assay is partially purified Factor VIII:C/vWF which has been standardized against a conventional method (electroimmunoassay). The results show a close correlation (correlation coefficient 0.956) as compared to the standard Laurell electroimmunoassay technique. Inter-assay and intra-assay coefficients of variation were less than 5%. The technique is simple to perform and results may be obtained within three hours of specimen collection.