A new in vitro system for the detection of platelet dysfunction, PFA-100™* has been developed. It provides a quantitative measure of platelet function in anticoagulated whole blood. The system comprises a microprocessor-controlled instrument and a disposable test cartridge containing a biologically active membrane. The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into the membrane. The membrane is coated with collagen and epinephrine or adenosine 5’-diphosphate. The presence of these biochemical stimuli, and the high shear rates generated under the standardized flow conditions, result in platelet attachment, activation, and aggregation, slowly building a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the m“closure time.” We have found that impairment of von Willebrand factor, or inhibition of platelet receptors glycoprotein Ib or IIblIIIa with monoclonal antibodies or peptides, resulted in abnormal closure times. An antifibrinogen antibody, in contrast, failed to show any effect. The test appears to be sensitive to platelet adherence and aggregation abnormalities. The PFA-100™ system has potential applications in routine evaluation of platelet function in the clinical setting because of its accuracy, ease of operation, and rapid turnaround of results. * Under evaluation
Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M. L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE,-treated (M. L.) platelets, ADP was without effect. The binding of [3H12-methylthio-ADP de-creased from 836±126 molecules/platelet for normals to 30±17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M. L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP HIb-Ma complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shapechanged platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP. (J. Clin. Invest. 1995. 95:1612-1622
A new in vitro system for the evaluation of platelet function, PFA-100 (currently under evaluation) is characterized. The system monitors platelet aggrega tion on a collagen-epinephrine-coated membrane as whole blood sample is aspirated under controlled flow conditions through a microscopic aperture cut into the membrane. The time required for the platelet plug to oc clude the aperture (closure time) is indicative of the plate let function in the sample. Incubation of normal blood samples with antibodies to glycoprotein (GP) Ib, GPIIb- IIIa, von Willebrand factor, or with the peptide Arg-Gly- Asp-Ser, resulted in a concentration-dependent prolonga tion in closure time. An anti-fibrinogen antibody did not impact the closure time. Closure time was also prolonged when the hematocrit or the platelet count was lowered to pathological values. The study indicates that PFA-100 could detect defects in platelet adhesion or aggregation. The simplicity of the test makes PFA-100 unique for rapid screening of a variety of platelet dysfunction.
Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin- stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin- stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while immunogold staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and immunogold staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb- IIIa complexes and of the alpha-granule membrane GP, GMP-140.
The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 +/- 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either 111Indium-oxine or 51Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.
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